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Dopamine but not L-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson's and other dopamine deficiency states
Abstract
Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50 μM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50 μM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50 μM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.
... An isocratic, reverse-phase HPLC coupled to an electrochemical detector method, adapted from Allen et al. (2013), was used to measure GSH levels in cell lysate. 15 mM HPLC grade o-phosphoric acid was used as the mobile phase with a flow rate of 0.5 ml/min. ...
... In this study, cells were exposed to L-DOPA for a relatively short period of time (1 and 3 hours) which may not have been sufficient to be detrimental to the cells. Allen and colleagues (2013) have shown that 50 µM L-DOPA increased GSH levels but only after 24 hours (Allen et al. 2013). Similar findings were documented in rat PC12 neuronal cells, where cellular toxicity of L-DOPA was documented only after 24-48 hours (Jin et al. 2008). ...
... In addition, SH-SY5Y cells were used to investigate the effect of heterozygous mutations in GBA on mitochondrial function(Li et al. 2019).Finally, PD is a disease of cellular oxidative stress imbalance. SH-SY5Y cells were used to study the effect of dopamine and L-DOPA oxidation and rotenone toxicity on the cellular GSH levels, the major antioxidant in the cell(Allen et al. 2013;Watabe and Nakaki 2008).Despite the wide use of this cell line, the literature is not clear on whether SH-SY5Y cells can be used in their proliferative state or whether differentiation is necessary. Proliferative SH-SY5Y cells form a heterogenous cell population of constantly dividing cells that are non-polarised and have few neuronal processes, making them dissimilar to post-mitotic neurons. ...
Parkinson’s disease (PD) is the second most common neurodegenerative disorder. The exact molecular mechanism of disease remains unclear. Several factors are proposed to play part including, but not limited to, decreased activity of mitochondrial complex I and lysosomal glucocerebrosidase enzymes and disrupted cellular antioxidant defence and lysosomal acidification. In addition, there is growing support for a role of organelle crosstalk between mitochondria and lysosome, the disruption of which is proposed to play part in PD pathology. The nature and consequence of this crosstalk remains unclear. The SH-SY5Y neuronal cell line model is commonly used to investigate PD mechanisms and potential therapeutics. However, functional analysis of the suitability of the cell line in its proliferative state or the necessity for differentiation remains unclear. Furthermore, iPSC-derived dopaminergic neurons are another commonly used model for PD and related diseases however, validating their functional dopamine metabolism is important to determine disease mechanism and test potential therapeutics. In this thesis, a host of biochemical tools, including HPLC measurement of neurotransmitter metabolites and enzyme activity assays, were used to elucidate the aforementioned ambiguities. The findings demonstrate that although there are similarities between proliferative and differentiated phenotypes of SH-SY5Y cells, there are also significant differences. Notably, the rate of dopamine turnover and the activity of lysosomal glucocerebrosidase were significantly higher in differentiated SH-SY5Y cells. In contrast, mitochondrial electron transport chain complexes’ activities were similar between the two phenotypes, despite a significant difference in mitochondrial content. Therefore, care should be taken when choosing either phenotype as a PD model. In addition, 4the findings demonstrate that inhibition of either mitochondrial complex I or lysosomal glucocerebrosidase affect both the ratio of pro-cathepsin D/cathepsin D protein expression and enzyme activity. Cathepsin D is one of the most ubiquitous lysosomal enzymes, the state of which can be used as reflection of the degree of lysosomal acidification. This shines a light on the potential involvement of both lysosomal glucocerebrosidase and mitochondrial complex I in maintenance of lysosomal acidification. This could be a consequence of a more dynamic crosstalk between mitochondria and lysosomes than previously thought. Moreover, the work presented provides a method for validation of the dysfunctional dopamine metabolism in iPSC derived dopaminergic neuronal disease models for aromatic amino acid decarboxylase deficiency and PD patients carrying mutations in PINK1. In addition, it provides a proof of concept for the effectiveness of both lentivirus-based gene therapy and levodopa treatment to restore dopamine metabolism in aromatic amino acid decarboxylase deficiency.
... Cells were seeded in two 25 cm 2 flasks per experiment as previously described in section 2.2.1. On day 6 after seeding, one flask was treated with 10 μM NSD-1015 and the other one was not treated and used as a control (Allen et al., 2013). 24 h later, the medium was removed and cells were treated with 100 μM L-DOPA for 1 h as described in section 2.2.2.1. ...
... To confirm whether L-DOPA was being metabolised via the dopamine pathway, AADC was inhibited before L-DOPA incubation. SH-SY5Y cells were treated with 10 µM NSD-1015 for 24 h (Allen et al., 2013), followed by 100 µM L-DOPA incubation for 1 h. While no effect was observed on 3-OMD release, NSD-1015 decreased the concentration of the molecules downstream in the pathway compared to the control (Figure 4.3). ...
... GSH has been described as essential to prevent mitochondrial damage by peroxynitrite and to carry out aminochrome excretion (Munoz et al., 2012;Heales et al., 1995). In the current study, GSH was detected in SH-SY5Y cells, although the values obtained were lower than the ones previously published in the literature (Allen et al., 2013). This could be due to the differences in the cell culture length or cell passage. ...
Parkinson’s disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic neurons in the substantia nigra. Different pathogenic mechanisms have been implicated, including loss of mitochondrial complex I function and dysfunction of lysosomal glucocerebrosidase (GBA1) (Neumann et al., 2009; Schapira et al., 1990). Also, it has been hypothesised that serotonin metabolism could be affected in these patients due to the number of enzymes shared by both pathways (Albizu et al., 2011). This thesis considers the potential involvement of complex I and GBA1 in PD using HPLC analysis of changes in the extracellular levels of the metabolites of dopamine and serotonin, and the expression and activity of the enzymes of the dopamine pathway. Using SH-SY5Y cells, complex I deficiency was modelled using rotenone, and GBA1 deficiency was modelled using conduritol B epoxide (CBE). Inhibition of mitochondrial complex I or GBA1 significantly increased extracellular concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA), direct products of the degradation by monoamine oxidase (MAO) of dopamine and serotonin respectively. These results suggest increased MAO activity, providing evidence for the involvement of impaired complex I or GBA1 activity in the dopamine deficiency seen in PD. As MAO produces hydrogen peroxide as a side-product, its increased activity could enhance the oxidative stress present in PD (Dias et al., 2013). Therefore, intracellular GSH levels were quantified to determine whether the antioxidant mechanisms were affected, but no changes were observed. In addition to the main project, I collaborated with a number of groups to study monoamine metabolism in parkinsonian models. Also, the glycoprofile of cerebrospinal fluid (CSF) of patients with and without impaired dopamine metabolism was studied to explore the possibility of using glycans as pathologic biomarkers.
... Additionally, SH-SY5Y cells were used to study PD related lysosomal dysfunction in terms of effect of lysosomal GBA inhibition on dopamine and serotonin metabolism (de la Fuente et al., 2017), effect of heterozygous GBA mutations on mitochondrial function (Li et al., 2019) and effect of GBA inhibition on mitochondrial function and oxidative stress (Cleeter et al., 2013). Finally, SH-SY5Y cells have been employed to assess the glutathione system in response to dopamine and L-DOPA induced oxidative stress (Allen et al., 2013) and effect of the neurotoxin rotenone on GSH levels (Watabe and Nakaki, 2008). ...
... Glutathione levels in cell lysate was measure by a method adapted from (Allen et al., 2013). ...
Parkinson's disease is a multifactorial neurodegenerative disease. The cellular pathology includes dopamine depletion, decrease in mitochondrial complex I enzyme activity, lysosomal glucocerebrosidase enzyme activity and glutathione levels. The SH-SY5Y human neuroblastoma cell line is one of the most widely used cell line model for Parkinson's disease. However, the consensus on its suitability as a model in its proliferative or differentiated state is lacking. In this study, we characterized and compared the biochemical processes most often studied in PD. This in proliferative and differentiated phenotypes of SH-SY5Y cells and several differences were found. Most notably, extracellular dopamine metabolism was significantly higher in differentiated SH-SY5Y. Furthermore, there was a greater variability in glutathione levels in proliferative phenotype (+/- 49%) compared to differentiated (+/- 16%). Finally, enzyme activity assay revealed significant increase in the lysosomal enzyme glucocerebrosidase activity in differentiated phenotype. In contrast, our study has found similarities between the two phenotypes in mitochondrial electron transport chain activity and tyrosine hydroxylase protein expression. The results of this study demonstrate that despite coming from the same cell line, these cells possess some key differences in their biochemistry. This highlights the importance of careful characterization of relevant disease pathways to assess the suitability of cell lines, such as SH-SY5Y cells, for modelling PD or other diseases, i.e. when using the same cell line but different differentiation states.
... Not even in vivo studies have provided convincing data on the toxicity of L-dopa and DA ( Mytilineou et al. 2003;Schapira 2008) even though both molecules have demonstrated protective effects at sub-toxic concentrations (Allen et al. 2013;Cornetta et al. 2009;Prigione et al. 2006). Furthermore, it must be considered that L-dopa is rapidly metabolised by catechol-o-methyl transferase (COMT) and aromatic L-amino acid decarboxylase (AADC) present both in peripheral districts and in the brain ( Fox and Lang 2008;Olanow et al. 2009). ...
... Despite the fact that the oxidation of L-dopa (and DA) might produce ROS ( Allen et al. 2013;Lai and Yu 1997), we demonstrate that L-dopa has a double nature: it behaves both as an antioxidant molecule, increasing the expression of CAT and GPX-3, and as a scavenger molecule in vitro. These effects are evident at doses that are higher than those that are physiologically present in the plasma of patients receiving pharmacological therapy. ...
The main pathochemical hallmark of Parkinson's disease (PD) is the loss of dopamine in the striatum of the brain, and the oral administration of levodopa (L-dopa) is a treatment that partially restores the dopaminergic transmission. In vitro assays have demonstrated both toxic and protective effects of L-dopa on dopaminergic cells, while in vivo studies have not provided any convincing data. The peripheral metabolic pathways significantly decrease the amount of L-dopa reaching the brain; therefore, all of the current commercial formulations require an association with an inhibitor of dopa-decarboxylase, such as carbidopa. However, the dosage and the actual effectiveness of carbidopa have not yet been well defined. PD patients exhibit a reduced efficiency of the endogenous antioxidant system, and peripheral blood lymphocytes (PBLs) represent a dopaminergic system for use as a cellular model to study the pharmacological treatments of neurodegenerative disorders in addition to analysing the systemic oxidative stress. According to our previous studies demonstrating a protective effect of both L-dopa and carbidopa on neuroblastoma cells in vitro, we used the PBLs of healthy donors to evaluate the modulation of DNA damage by different concentrations of L-dopa and carbidopa in the presence of oxidative stress that was exogenously induced by H2O2. We utilised a TAS assay to evaluate the in vitro direct scavenging activity of L-dopa and carbidopa and analysed the expression of genes that were involved in cellular oxidative metabolism. Our data demonstrate the antioxidant capacity of L-dopa and carbidopa and their ability to protect DNA against oxidative-induced damage that derives from different mechanisms of action.
... Although endogenous dopamine levels could be detected in hDANs derived from much larger cell volumes, we employed 50µM L-DOPA treatment overnight (16h) to enhance dopamine metabolism without risk of dopamine toxicity (Allen et al., 2013, Burbulla et al., 2017. Following detachment of hDANs with Accumax, the cell suspensions were washed in PBS and cells counted. ...
PINK1 loss-of-function mutations cause early onset Parkinson disease. PINK1-Parkin mediated mitophagy has been well studied, but the relevance of the endogenous process in the brain is debated.
Here, the absence of PINK1 in human dopaminergic neurons inhibits ionophore-induced mitophagy and reduces mitochondrial membrane potential. Compensatory, mitochondrial renewal maintains mitochondrial morphology and protects the respiratory chain. This is paralleled by metabolic changes, including inhibition of the TCA cycle enzyme mAconitase, accumulation of NAD⁺, and metabolite depletion. Loss of PINK1 disrupts dopamine metabolism by critically affecting its synthesis and uptake. The mechanism involves steering of key amino acids toward energy production rather than neurotransmitter metabolism and involves cofactors related to the vitamin B6 salvage pathway identified using unbiased multi-omics approaches.
We propose that reduction of mitochondrial membrane potential that cannot be controlled by PINK1 signaling initiates metabolic compensation that has neurometabolic consequences relevant to Parkinson disease.
... The reaction between DA-o-Q and GSH leads to the formation of the 5-S-glutathionyl-dopamine (5-S-GSH-DA) conjugate, as the main product (Bisaglia et al., 2010) (See Fig. 2. Reaction 1). However, experimental studies have shown that 5-S-GSH-DA is rapidly catabolized by the enzymes gamma-glutamyl transferase (GGT) and dipeptidases, resulting in the formation of 5-S-Cys-DA (Allen et al., 2013;Dickinson and Forman, 2002). Furthermore, several studies have reported the increased activity of GGT in brain samples of PD patients, which might support its critical role in the conversion of 5-S-GSH-DA into 5-S-Cys-DA (Dagnino-Subiabre et al., 2000;Sagara et al., 1993). ...
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and is characterized for being an idiopathic and multifactorial disease. Extensive research has been conducted to explain the origin of the disease, but it still remains elusive. It is well known that dopamine oxidation, through the endogenous formation of toxic metabolites, is a key process in the activation of a cascade of molecular events that leads to cellular death in the hallmark of PD. Thio-catecholamines, such as 5-S-cysteinyl-dopamine, 5-S-glutathionyl-dopamine and derived benzothiazines, are endogenous metabolites formed in the dopamine oxidative degradation pathway. Those metabolites have been shown to be highly toxic to neurons in the substantia nigra pars compacta, activating molecular mechanisms that ultimately lead to neuronal death. In this review we describe the origin, formation and the toxic effects of 5-S-cysteinyl-dopamine and its oxidative derivatives that cause death to dopaminergic neurons. Furthermore, we correlate the formation of those metabolites with the neurodegeneration progress in PD. In addition, we present the reported neuroprotective strategies of products that protect against the cellular damage of those thio-catecholamines. Finally, we discuss the advantages in the use of 5-S-cysteinyl-dopamine as a potential biomarker for PD.
... One limitation of our study may be the concentration of DA. Although the exact concentration of DA in the brain is not known, 50 mmol/L of DA that was used in the past reports (Allen et al., 2013) and 100 mmol/L of DA in this study might be too high. ...
Oxidative stress plays an important role in the onset and progression of Parkinson disease. Although released dopamine at the synaptic terminal is mostly reabsorbed by dopaminergic neurons, some dopamine is presumably taken up by astroglia. This study examined the dopamine-induced astroglial protective function through the activation of the pentose-phosphate pathway (PPP) to reduce reactive oxygen species (ROS). In vitro experiments were performed using striatal neurons and cortical or striatal astroglia prepared from Sprague-Dawley rats or C57BL/6 mice. The rates of glucose phosphorylation in astroglia were evaluated using the [¹⁴C]deoxyglucose method. PPP activity was measured using [1-¹⁴C]glucose and [6-¹⁴C]glucose after acute (60 min) or chronic (15 hr) exposure to dopamine. ROS production was measured using 2′,7′-dichlorodihydrofluorescein diacetate. The involvement of the Kelch-like ECH-associated protein 1 (Keap1) or nuclear factor-erythroid-2-related factor 2 (Nrf2) system was evaluated using Nrf2 gene knockout mice, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction analysis for heme oxygenase-1. Acute exposure to dopamine elicited increases in astroglial glucose consumption with lactate release. PPP activity in astroglia was robustly enhanced independently of Na⁺-dependent monoamine transporters. In contrast, chronic exposure to dopamine induced moderate increases in PPP activity via the Keap1/Nrf2 system. ROS production from dopamine increased gradually over 12 hr. Dopamine induced neuronal cell damage that was prevented by coculturing with astroglia but not with Nrf2-deficient astroglia. Dopamine-enhanced astroglial PPP activity in both acute and chronic manners may possibly reduce neuronal oxidative stress.
... The most common applications for the methods described here include the diagnosis of primary genetic conditions that lead to neurotransmitter deficiencies and other severe early onset neurological diseases that can be associated with disturbances in brain dopamine and serotonin availability 10,11 . The procedures may also be suitable for the quantification of monoamines, such as dopamine, serotonin and related compounds, in experimental animal and cellular models after adequate sample extraction and purification steps, (acid precipitation on ice, centrifugation, and supernatant filtration) as previously reported 12,13,14 . Brain microdialysis procedures also allow the determination of monoamines and other molecules in interstitial tissue fluid 15 . ...
The presence of monoamines and their cofactors (the pterins and vitamin B6 (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors. This protocol describes step-by-step procedures for CSF sample preparation for the analysis of different molecules, HPLC calibration and analysis, and data quantification and interpretation. Unlike plasma/tissue/blood samples, CSF requires minimal sample preparation: in this protocol, only the analysis of PLP requires mixing with trichloroacetic acid to release the protein-bound vitamin, centrifugation, and mixing of the supernatant with phosphate buffer and sodium cyanide for derivatization in alkaline conditions. Monoamines are analyzed by HPLC with coulometric electrochemical detection (ED), pterins are analyzed by HPLC with coupled coulometric electrochemical and fluorescence detection, and PLP is analyzed by HPLC with fluorescence detection. The quantification of all compounds is achieved by external calibration procedures, and internal quality control and standards are analyzed in each run. We anticipate that investigation of dopamine and serotonin disturbances will be facilitated by measurements of these compounds in human CSF and other biological samples. The estimated time for the different procedures primarily depends on the electrochemical detector stabilization. Overnight stabilization of this detector is advised, and, after that step, preanalytical equilibration rarely exceeds 3 h.
... It is believed that low serotonin is one of the factors which are responsible for depressive symptoms in schizophrenic patients. 7,8 It has been reported that the consumption of tryptophan, as a precursor of serotonin, can lead to diminished depression. 9 Therefore, scientists have to find other ways to increase neurotrophic factors in human brain. ...
Neurotransmitters and neurotrophic factors are signaling molecules that play a crucial role in cell proliferation, differentiation, survival and functions of neurons. It is believed that caloric restriction could help the health of the nervous system by affecting the synthesis of neurotrophins and neurotransmitter and oxygen radical metabolism. The objective was to investigate the plasma levels of serotonin, dopamine, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) in 29 healthy fasted subjects (22 women and 7 men) during the month of fasting in Ramadan. The levels of these factors were measured (using ELISA method) three times, 2 days before the fasting month as a control, on the 14th and 29th day of Ramadan as test groups. In addition, these factors were investigated in the group of women only. According to our investigation, the plasma levels of serotonin, BDNF and NGF were significantly increased during fasting month of Ramadan. In detail, the levels of these factors were increased in 14th and 29th day test groups compared to controls (P<0.05). Moreover, these levels were significantly increased on the 29th day compared to the 14th day test groups, but there were no differences between dopamine levels in all groups. Furthermore, the results obtained in women’s groups were the same as those obtained in previous groups. Our findings suggest that plasma levels of serotonin, BDNF and NGF were significantly increased during fasting month of Ramadan.
... Dopamine, 3-OMD, HVA, DOPAC and 5-HIAA were quantified using reverse-phase HPLC with an electrochemical detector, following the method of Allen et al. (2013) with some modifications for the contemporary detection of all five compounds. The stationary phase was maintained at 27°C. ...
Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic and serotoninergic signalling. A number of pathogenic mechanisms have been implicated including loss of mitochondrial function at the level of complex I, and lysosomal metabolism at the level of lysosomal glucocerebrosidase (GBA1). In order to investigate further the potential involvement of complex I and GBA1 in PD, we assessed the impact of loss of respective enzyme activities upon dopamine and serotonin turnover. Using SH-SY5Y cells, complex I deficiency was modelled by using rotenone whilst GBA1 deficiency was modelled by the use of conduritol B epoxide (CBE). Dopamine, its principal metabolites, and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the extracellular medium were quantified by HPLC. Inhibition of complex I significantly increased extracellular concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-HIAA. Comparable results were observed with CBE. These results suggest increased monoamine oxidase activity and provide evidence for involvement of impaired complex I or GBA1 activity in the dopamine/serotonin deficiency seen in PD. Use of extracellular media may also permit relatively rapid assessment of dopamine/serotonin metabolism and permit screening of novel therapeutic agents.
... This change is due to an increase in the intracellular dopamine metabolism that is dependent on monoamine oxidase and the auto-oxidation of L-DOPA and dopamine to quinones (Basma, Morris, Nicklas, & Geller, 1995; for review see Kostrzewa, Kostrzewa, & Brus, 2002). Dopamine, but not L-DOPA, stimulates neuronal glutathione metabolism (Allen, Ullah, Hargreaves, Land, & Heales, 2013), an enzyme that acts as a major cellular antioxidant (Han, Mytilineou, & Cohen, 1996). Likewise, a significant increase in cysteinyl adducts of L-DOPA and dopamine has been found in the substantia nigra of PD patients, suggesting the cytotoxic nature of dopamine oxidation (Spencer et al., 1998). ...
Inflammation in Parkinson's disease (PD) is a new concept that has gained ground due to the potential of mitigating dopaminergic neuron death by decreasing inflammation. The solution to this question is likely to be complex. We propose here that the significance of inflammation in PD may go beyond the nigral cell death. The pathological process that underlies PD requires years to reach its full extent. A growing body of evidence has been accumulated on the presence of multiple inflammatory signs in the brain of PD patients even in very late stages of the disease. Because neuron-microglia-astrocyte interactions play a major role in the plasticity of neuronal response to L-DOPA in post-synaptic neurons, we focused this review on our recent results of L-DOPA-induced dyskinesia in rodents correlating it to significant findings regarding glial cells and neuroinflammation. We showed that in the rat model of PD/L-DOPA-induced dyskinesia there was an increased expression of inflammatory markers, such as the enzymes COX2 in neurons and iNOS in glial cells, in the dopamine-denervated striatum. The gliosis commonly seem in PD was associated with modifications in astrocytes and microglia that occur after chronic treatment with L-DOPA. Either as a cause, consequence, or promoter of progression of neuronal degeneration, inflammation plays a role in PD. The key aims of current PD research ought to be to elucidate (i) the time sequence in which the inflammatory factors act in PD patient brain and (ii) the mechanisms by which neuroinflammatory response contributes to the collateral effects of L-DOPA treatment. This article is protected by copyright. All rights reserved.
... Accordingly, long term studies in PD described a lower advance rate with dopamine agonist treatment alone compared with L-Dopa monotherapy, employing functional imaging techniques of the dopaminergic nigrostriatal system as biological marker for the progression of PD (Whone et al. 2003;Parkinson Study Group 2002). Limitations of this pilot trial are the missing direct GSH determination due to technical reasons (Allen et al. 2013) and that this investigation was not done in the central nervous system. Moreover we only provide changes of the optical density values in the performed ELISA technique, because the used aliquots for the measurement of the baseline and 60 min values result from the same investigation day on the one hand but were stored over different intervals by -80°C on the other hand. ...
Exposure to free radicals influences synthesis, degradation and function of proteins, such as repulsive guidance molecule A. Decay of this protein is essential for neuronal maintenance and recovery. Levodopa elevates oxidative stress. Therefore levodopa may impact repulsive guidance molecule A metabolism. Objectives were to investigate plasma concentrations of repulsive guidance molecule A, levodopa, cysteine and cysteinyl-glycine before and 1 h after levodopa application in patients with Parkinson's disease. Cysteine and cysteinyl-glycine as biomarkers for oxidative stress exposure decreased, repulsive guidance molecule A and levodopa rose. Repulsive guidance molecule A remained unchanged in levodopa naïve patients, but particularly went up in patients on a prior chronic levodopa regimen. Decay of cysteine specifically cysteinyl-glycine results from an elevated glutathione generation with rising cysteine consumption respectively from the alternative glutathione transformation to its oxidized form glutathione disulfide after free radical scavenging. Repulsive guidance molecule A rise may inhibit physiologic mechanisms for neuronal survival.
... However, more permeable GSH precursors, such as glutamyl cysteine ethyl ester (GCEE) and glutathione ethyl ester (GEE) are interesting prospects for future clinical investigations [150,151]. In addition, recent studies showed that dopamine, but not L-DOPA, enhanced GSH release from astrocytes and trafficking to neurons [152], while the antiepileptic drug zonisamide increased GSH production by astrocytes, reduced -synuclein neuro-toxicity and protected dopaminergic neurons in a mouse model of PD [153,154]. Zonisamide is currently in phase II clinical trial for PD [155]. ...
The causes of neurodegenerative disorders are multiple, and for most of them a mechanistic understanding is still lacking. However, neurodegenerative diseases such as Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD) all share common features that include elevated oxidative stress levels and impaired energy metabolism in the nervous system. Most of the current treatments are only successful at alleviating some of the pathological symptoms, but fail at preventing neurodegeneration. There is therefore an urgent need for innovative and more efficient treatments for neurodegenerative disorders. We review here the central role played by astrocytes in the regulation of brain homeostasis, protection and function by supporting neuronal health and activity. In particular, astrocytes are key partners of neuronal metabolism, notably through activation of the astrocyte-neuron lactate shuttle (ANLS). They also control the levels of extracellular glutamate, production of antioxidant molecules, disposal of neuronal waste products, storage of energy in the form of glycogen, and expression of neurotrophic factors. These mechanisms, which are key for brain activity and cognition, also largely contribute to neuronal degeneration in pathological situations. Thus, as astrocytes appear to play a key role in the etiology of neurodegenerative disorders, a growing interest has arisen for astrocyte-mediated pathways as targets for drugs that aim at treating the root causes of the pathology. We present here the most recent and promising astrocyte-based therapeutic approaches - from fundamental discoveries to clinical trials - that intent to sustain neuronal health and function in neurodegenerative disorders.
... GSH in the brain is responsible also for detoxication of the DA autooxidation product DA-O-quinone by forming a conjunct, 5-S-glutathionyldopamine (5-S-Glu-DA), which undergoes further metabolism end excretion [13,60]. The protective role of GSH to neurons subjected to excessive concentrations of DA has been shown by Allen et al. [61]. In this study, the increased synthesis of GSH was induced by administration of DA alone, as well as L-dopa, while AADC remained active. ...
Parkinson's disease (PD) is one of the most common neurological diseases in elderly people. The mean age of onset is 55 years of age, and the risk for developing PD increases 5-fold by the age of 70. In PD, there is impairment in both motor and nonmotor (NMS) functions. The strategy of PD motor dysfunction treatment is simple and generally based on the enhancement of dopaminergic transmission by means of the L-dihydroxyphenylalanine (L-dopa) and dopamine (DA) agonists. L-dopa was discovered in the early -60's of the last century by Hornykiewicz and used for the treatment of patients with PD. L-dopa treatment in PD is related to decreased levels of the neurotransmitter (DA) in striatum and ab-sence of DA transporters on the nerve terminals in the brain. L-dopa may also indirectly stimulate the receptors of the D1 and D2 families. Administration of L-dopa to PD patients, especially long-time therapy, may cause side effects in the form of increased toxicity and inflammatory response, as well as disturbances in biothiols metabolism. Therefore, in PD pa-tients treated with L-dopa, monitoring of oxidative stress markers (8-oxo-2'-deoxyguanosine, apoptotic proteins) and in-flammatory factors (high-sensitivity C-reactive protein, soluble intracellular adhesion molecule), as well as biothiol com-pounds (homocysteine, cysteine, glutathione) is recommended. Administration of vitamins B6, B12, and folates along with an effective therapy with antioxidants and/or anti-inflammatory drugs at an early stage of PD might contribute to improvement in the quality of the life of patients with PD and to slowing down or stopping the progression of the disease.
... Cysteine is one essential component for GSH synthesis (Smeyne and Smeyne 2013). The observed decline of both substrates supports the hypothesis that both GSH consumption and synthesis probably went up for compensation of an increased free radical generation after L-dopa intake Muhlack 2011, 2012;Allen et al. 2013). There was no pronounced impact of additional catechol-O-methyltransferase inhibition on the Cys-Gly, respectively, cysteine plasma occurrence. ...
Oxidative stress is influenced by the thiol homeostasis, which regulates the redox milieu via glutathione. Components of glutathione metabolism are cysteine and cysteinyl-glycine. Both substrates decay following levodopa application or dopamine-related oxidative stress. Objective was to investigate the impact of an acute levodopa application with and without catechol-O-methyltransferase inhibitor on cysteine- and cysteinyl-glycine plasma levels. On two investigation days, 13 patients with Parkinson's disease took one retarded release 200-mg levodopa/50 mg carbidopa-containing tablet or one 150-mg levodopa/50-mg carbidopa/200-mg entacapone formulation under standardized conditions. Levodopa, 3-O-methyldopa, cysteine and cysteinyl-glycine were measured at baseline, 80 and 140 min following levodopa administration. Cysteine and cysteinyl-glycine similarly decreased, levodopa was nearly equal during both conditions. Entacapone lowered 3-O-methyldopa. Cysteine decay may be due to an elevated glutathione generation, which consumes cysteine. Cysteinyl-glycine decrease results from the alternative glutathione transformation to its oxidized form glutathione dissulfide after free radical scavenging.
... Blocking an enzyme in the dopamine synthesis pathway prevented increase in glutathione levels in SHSY5Y cells. Dopamine may upregulate transcription of genes involved in glutathione synthesis and release [19]. Antioxidant activity is also provided by 4 ...
The cell has an intricate quality control system to protect its mitochondria from oxidative stress. This surveillance system is multi-tiered and comprises molecules that are present inside the mitochondria, in the cytosol, and in other organelles like the nucleus and endoplasmic reticulum. These molecules cross talk with each other and protect the mitochondria from oxidative stress. Oxidative stress is a fundamental part of early disease pathogenesis of
neurodegenerative diseases. These disorders also damage the cellular quality control machinery that protects the cell against oxidative stress. This exacerbates
the oxidative damage and causes extensive neuronal cell death that is characteristic of neurodegeneration.
Aromatic l-amino acid decarboxylase deficiency (AADC-DY) is caused by one or more mutations in the DDC gene, resulting in the deficit in catecholamines and serotonin neurotransmitters. The disease has limited therapeutic options with relatively poor clinical outcomes. Accumulated evidence suggests the involvement of neurodegenerative mechanisms in the etiology of AADC-DY. In the absence of neurotransmitters’ neuroprotective effects, the accumulation and the chronic presence of several neurotoxic metabolites including 4-dihydroxy-L-phenylalanine, 3-methyldopa, and homocysteine, in the brain of subjects with AADC-DY, promote oxidative stress and reduce the cellular antioxidant and methylation capacities, leading to glial activation and mitochondrial dysfunction, culminating to neuronal injury and death. These pathophysiological processes have the potential to hinder the clinical efficacy of treatments aimed at increasing neurotransmitters’ synthesis and or function. This review describes in detail the mechanisms involved in AADC-DY neurodegenerative etiology, highlighting the close similarities with those involved in other neurodegenerative diseases. We then offer novel strategies for the treatment of the disease with the objective to either reduce the level of the metabolites or counteract their prooxidant and neurotoxic effects. These treatment modalities used singly or in combination, early in the course of the disease, will minimize neuronal injury, preserving the functional integrity of neurons, hence improving the clinical outcomes of both conventional and unconventional interventions in AADC-DY. These modalities may not be limited to AADC-DY but also to other metabolic disorders where a specific mutation leads to the accumulation of prooxidant and neurotoxic metabolites.
Parkinson’s disease (PD) is a progressive chronic neurodegenerative condition characterized by the loss of dopaminergic neurons within the substantia nigra. Current PD therapeutic strategies are mainly symptomatic and can lead to motor complications overtime. As a result, alternative medicine may provide an effective adjuvant treatment for PD as an addition to or as a replacement of the conventional therapies. The aim of this work was to evaluate the effects of Bee Venom (BV) and dopamine (DA)-loaded nanoparticles in a reserpine-induced animal model of PD. After inducing PD with reserpine injection, different groups of male rats were treated with L-Dopa, BV, DA-nanoparticles. Our findings showed that BV and DA-nanoparticles administration restored monoamines, balanced glutamate/GABA levels, halted DNA fragmentation, decreased pro-inflammatory mediators (IL-1β and TNF-α), and elevated anti-inflammatory mediators (PON1) and neurotropic factor (BDNF) levels in comparison with conventional therapy of PD. Furthermore, in a reserpine-induced PD rat model, the ameliorative effects of BV were significantly superior to that of DA-nanoparticles. These findings imply that BV and DA-nanoparticles could be useful as adjuvant treatments for PD.
In this study, we report the synthesis of self-assembled dityrosine nanotubes as a biologically functional scaffold and their interactions with neural cells. Quantum chemical methods were used to determine the forces involved in the self-assembly process. The physicochemical properties of the nanostructures relevant to their potential as bioactive scaffolds were characterized. The morphology, secondary structure, crystallinity, mechanical properties, and thermal characteristics of YY nanotubes were analyzed. The influence of these nanotubes as scaffolds for neural cells was studied in vitro to understand their effects on cell proliferation, morphology, and gene expression. The scanning electron microscopy and fluorescence confocal microscopy demonstrated the feasibility of nanotube scaffolds for enhanced adhesion to rat and human neural cells (PC12 and SH-SY5Y). Preliminary ELISA and qPCR analyses demonstrate the upregulation of dopamine synthesis and genes involved in dopamine expression and differentiation. The expression levels of DβH, AADC, VMAT2 and MAOA in SH-SY5Y cells cultured on the nanotube scaffolds for 7 days were elevated in comparison to the control cells.
In Parkinson's disease, current predominant treatment with intermittent oral administration of levodopa (l-dopa) remains the reference standard, but it has pharmacological drawbacks that trigger motor fluctuations and dyskinesia. To overcome these challenges, we demonstrate greater efficacy in either an acute 1-methyl-4-phenyl-1,2,3,6-terahydropyridine (MPTP) or chronic 6-hydroxydopamine (6-OHDA) lesioning model when dopamine is prepared under anaerobic (A-dopamine) conditions and continually administered intracerebroventricularly proximal to the striatum. This regimen was compared with dopamine prepared aerobically and administered by the same route, as well as with conventional peripheral l-dopa treatment. A-dopamine restored motor function and induced a dose-dependent increase in nigrostriatal dopaminergic neurons in MPTP mice. In the 6-OHDA rat model, circadianally administered A-dopamine improved motor activity without tachyphylaxia or dyskinesia. Indicative of a new therapeutic strategy for patients with l-dopa-related complications, continuous cerebral dosing of A-dopamine has greater efficacy over a large therapeutic index without undesirable side effects.
Mitochondrial dysfunction contributes to the pathogenesis of Parkinson’s disease but it is not clear why inherent mitochondrial defects lead specifically to the death of dopaminergic neurons of the mid brain. PINK1 is mitochondrial kinase and PINK1 mutations cause early onset Parkinson’s disease.
We found that in neuronal progenitors, PINK1 regulates mitochondrial morphology, mitochondrial contact to the endoplasmic reticulum (ER) and the phosphorylation of Miro1. A compensatory metabolic shift towards lipid synthesis provides mitochondria with the components needed for membrane renewal and oxidative phosphorylation, maintaining the mitochondrial network once mature.
Cholesterol is increased by loss of PINK1, promoting overall membrane rigidity. This alters the distribution of phosphorylated DAT at synapses and impairs dopamine uptake. PINK1 is required for the phosphorylation of tyrosine hydroxylase at Ser19, dopamine and calcium homeostasis and dopaminergic pacemaking.
We suggest a novel mechanism for PINK1 pathogenicity in Parkinson’s disease in addition to but not exclusive of mitophagy. We also provide a basis for potential therapeutics by showing that low doses of the cholesterol depleting drug ß-cyclodextrin reverse PINK1-specific phenotypes.
The scavenging rates of DOPA (dl- and l-3-(3,4-dihydroxyphenyl)alanine) and Tyr (tyrosine (dl- and l-3-(4-hydroxyphenyl)alanine)) against five reactive oxygen species (ROS) and methyl radical were measured with the use of electron spin resonance (ESR) spin-trapping method and the scavenging rate constants of DOPA and Tyr were determined. The scavenging rate constants for multiple active species increased in the order of O2(-)<RO<(1)O2<H3C<HO for Tyr and RO≈O2(-)<(1)O2≈H3C<HO for DOPA, and the differences in the radical scavenging abilities for l-enantiomers and dl-mixtures of DOPA and Tyr were shown. Further, based on the redox potentials, we have suggested that the primary chemical process of antioxidant reactions with O2(-) and (1)O2 can be characterized with the electron transfer of antioxidants (DOPA and Tyr).
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The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of
this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5′-
and 3′UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5′- and 3′UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either
a 5′UTR or a 3′UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine,
norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5′UTR of one of the most
abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3′UTR responded selectively to stimulation with
the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine.
We propose that BDNF mRNA variants represent “a quantitative code” for regulated expression of the protein. Thus, to discriminate
the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests.
Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and as a result, disturbances in GSH homeostasis are implicated in the etiology and/or progression of a number of human diseases, including cancer, diseases of aging, cystic fibrosis, and cardiovascular, inflammatory, immune, metabolic, and neurodegenerative diseases. Owing to the pleiotropic effects of GSH on cell functions, it has been quite difficult to define the role of GSH in the onset and/or the expression of human diseases, although significant progress is being made. GSH levels, turnover rates, and/or oxidation state can be compromised by inherited or acquired defects in the enzymes, transporters, signaling molecules, or transcription factors that are involved in its homeostasis, or from exposure to reactive chemicals or metabolic intermediates. GSH deficiency or a decrease in the GSH/glutathione disulfide ratio manifests itself largely through an increased susceptibility to oxidative stress, and the resulting damage is thought to be involved in diseases, such as cancer, Parkinson's disease, and Alzheimer's disease. In addition, imbalances in GSH levels affect immune system function, and are thought to play a role in the aging process. Just as low intracellular GSH levels decrease cellular antioxidant capacity, elevated GSH levels generally increase antioxidant capacity and resistance to oxidative stress, and this is observed in many cancer cells. The higher GSH levels in some tumor cells are also typically associated with higher levels of GSH-related enzymes and transporters. Although neither the mechanism nor the implications of these changes are well defined, the high GSH content makes cancer cells chemoresistant, which is a major factor that limits drug treatment. The present report highlights and integrates the growing connections between imbalances in GSH homeostasis and a multitude of human diseases.
Continuously cultured human neuroblastoma cell lines SK-N-SH, SK-N-BE(1), SK-N-BE(2), and SK-N-MC, as well as several clonally derived sublines, were assessed for their neurotransmitter characteristics. Two different methods were used: measurement of cholinergic and adrenergic enzyme activities and detection of neurotransmitters synthesized from radioactive precursors. Dopamine-β-hydroxylase (EC 1.14.2.1), choline acetyltransferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), and butyrylcholinesterase (EC 3.1.1.8) levels were compared to those of various normal and neuronal cell controls. The capacity of neuroblastoma lines and clones to convert radioactive tyrosine, choline, and glutamate to transmitter products was determined. SK-N-SH, SK-N-BE(1), and SK-N-BE(2) cells had moderately high levels of dopamine-β-hydroxylase activity. Whereas neuroblast-like clones of SK-N-SH had similar levels of this enzyme, epithelial-like clones had no detectable activity. SK-N-BE(2) cells showed the greatest capacity to convert tyrosine to dopamine. Although the SK-N-SH line itself was not tested, both neuroblast and epithelial-like clones, shown by karyotype analysis to have a common cell precursor, converted tyrosine to dopamine. In contrast, the SK-N-MC line and its clones appeared devoid of adrenergic properties. All cells tested except human fibroblast-like cells of normal origin were able to convert radioactive choline to acetylcholine. While all the cell lines had measurable choline acetyltransferase activity, levels were substantially higher (at least 4- to 12-fold) for SK-N-MC cells and its clonally derived subline MC-IXC. All cells tested converted glutamate to γ-aminobutyric acid. Results indicate that these human neuroblastoma cell lines have predominating neurotransmitter characteristics that are sometimes, but not always, expressed in clones derived from them. Since the SK-N-BE(1) and SK-N-BE(2) lines were isolated 5 months apart from the same patient and have similar activity levels of dopamine-β-hydroxylase, these characteristics may be stable and representative of the tumor cells in vivo. Furthermore, results obtained by the two different methodological approaches suggest that, whereas SK-N-MC cells show cholinergic and not adrenergic traits, SK-N-SH and possibly SK-N-BE(2) may be multipotential with regard to neuronal enzyme expression.
The ergot alkaloid [ 3H]dihydroergocryptine, a potent dopaminergic agonist, has been used to study binding sites in bovine anterior pituitary membranes. One function of the anterior pituitary gland, prolactin secretion, as we show is under typical dopaminergic control and was measured in vitro in another series of experiments using rat anterior pituitary cells in primary culture in order to establish a correlation between binding and a biological function. The dopaminergic specificity of ligand binding and the biological process was demonstrated by the fact that agonists competed for [ 3H]dihydroergocryptine binding and inhibited prolactin release with an identical order of potency: apomorphine > dopamine > epinephrine ≥ norepinephrine >> isoproterenol. Ergot alkaloids, which behave as agonists in this system, potently inhibited prolactin release from pituitary cells to about the same extent as dopamine (80 to 90% of basal levels) and were potent competitors of [ 3H]dihydroergocryptine binding (K(D) 0.2 to 0.5 nM). Dopaminergic antagonists competed for [ 3H]dihydroergocryptine binding in bovine anterior pituitary membranes in parallel to their ability to reverse either dopamine or dihydroergocornine inhibition of prolactin release from rat anterior pituitary cells. [ 3H]Dihydroergocryptine binding fulfilled another criterion of specific receptor sites in that binding to the anterior pituitary sites was saturable with an apparent dissociation constant (K(D)) of 2.2±0.6 nM and a mean saturation value of 0.32±0.02 pmol/mg of protein. The close correlation which exists between the properties of [ 3H]dihydroergocryptine binding to pituitary membrane receptors and characteristics of modulation of prolactin secretion by dopaminergic agents suggests that [ 3H]dihydroergocryptine binding sites are associated with mammotrophs and that these dopaminergic binding sites modulate prolactin secretion.
Aromatic L-amino acid decarboxylase (EC. 4.1.1.28) deficiency is a newly described inborn error of metabolism that affects serotonin and dopamine biosynthesis. The major biochemical markers for this disease are increases of L-dopa, 3-methoxytyrosine, and 5-hydroxytryptophan in urine, plasma, and cerebrospinal fluid together with decreased cerebrospinal fluid concentrations of homovanillic acid and 5-hydroxyindoleacetic acid. In addition, concentrations of vanillactic acid are increased in the urine. Specific HPLC and gas chromatography-mass spectrometry methods are described that permit the identification and measurement of these metabolites in the above body fluids. Simplified assays for human plasma L-dopa decarboxylase and liver L-dopa and 5-hydroxytryptophan decarboxylase, used to demonstrate the enzyme deficiency, are also reported.
We have characterized further the antioxidant responsive element (ARE) identified in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene and the NAD(P)H:quinone reductase gene by mutational and deletion analyses. Our data suggest that the sequence, 5'-puGTGACNNNGC-3' 3'-pyCACTGNNNCG-5' where N is any nucleotide, represents the core sequence of the ARE required for transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds (e.g. beta-naphthoflavone and 3-methylcholanthrene). We also have found that the ARE is responsive to hydrogen peroxide and phenolic antioxidants that undergo redox cycling. These latter data suggest that the ARE is responsive to reactive oxygen species and thus may represent part of a signal transduction pathway that allow eukaryotic cells to sense and respond to oxidative stress.
Glutathione, an essential cellular antioxidant required for mitochondrial function, is not synthesized by mitochondria but is imported from the cytosol. Rat liver mitochondria have a multicomponent system that underlies the remarkable ability of mitochondria to take up and retain glutathione. At external glutathione levels of less than 1 mM, glutathione is transported into the mitochondrial matrix by a high-affinity component (Km, approximately 60 microM; V max, approximately 0.5 nmol/min per mg of protein), which is saturated at levels of 1-2 mM and stimulated by ATP. Another component has lower affinity (Km, approximately 5.4 mM; Vmax, approximately 5.9 nmol/min per mg of protein) and is stimulated by ATP and ADP. Both components are inhibited by carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), glutamate, and ophthalmic acid. Increase of extramitochondrial glutathione promotes uptake and exchange; the intermembranous space seems to function as a recovery zone that promotes efficient recycling of matrix glutathione. The findings are in accord with in vivo data showing that (i) rapid exchange occurs between mitochondrial and cytosolic glutathione, (ii) lowering of cytosolic glutathione levels (produced by administration of buthionine sulfoximine) decreases export of glutathione from mitochondria to cytosol, and (iii) administration of glutathione esters increases glutathione levels in mitochondria more than those in the cytosol.
Deficiency of the antioxidant glutathione in brain appears to be connected with several diseases characterized by neuronal loss. To study neuronal glutathione metabolism and metabolic interactions between neurons and astrocytes in this respect, neuron-rich primary cultures and transient cocultures of neurons and astroglial cells were used. Coincubation of neurons with astroglial cells resulted within 24 hr of incubation in a neuronal glutathione content twice that of neurons incubated in the absence of astroglial cells. In cultured neurons, the availability of cysteine limited the cellular level of glutathione. During a 4 hr incubation in a minimal medium lacking all amino acids except cysteine, the amount of neuronal glutathione was doubled. Besides cysteine, also the dipeptides CysGly and gammaGluCys were able to serve as glutathione precursors and caused a concentration-dependent increase in glutathione content. Concentrations giving half-maximal effects were 5, 5, and 200 microM for cysteine, CysGly, and gammaGluCys, respectively. In the transient cocultures, the astroglia-mediated increase in neuronal glutathione was suppressed by acivicin, an inhibitor of the astroglial ectoenzyme gamma-glutamyl transpeptidase, which generates CysGly from glutathione. These data suggest the following metabolic interaction in glutathione metabolism of brain cells: the ectoenzyme gamma-glutamyl transpeptidase uses as substrate the glutathione released by astrocytes to generate the dipeptide CysGly that is subsequently used by neurons as precursor for glutathione synthesis.
Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) or pyrrolidine dithiocarbamate (PDTC) resulted in the up-regulation of the gamma-glutamylcysteine synthetase catalytic (GCS(h)) and regulatory (GCS(l)) subunit genes. Increased expression was associated with an increase in the binding of Nrf2 to electrophile response elements (EpRE) in the promoters of these genes. Nrf2 overexpression increased the activity of GCS(h) and GCS(l) promoter/reporter transgenes. Overexpression of an MafK dominant negative mutant decreased Nrf2 binding to GCS EpRE sequences, inhibited the inducible expression of GCS(h) and GCS(l) promoter/reporter transgenes, and reduced endogenous GCS gene induction. beta-NF and PDTC exposure also increased steady-state levels of MafG mRNA. In addition to Nrf2, small Maf and JunD proteins were detected in GCS(h)EpRE-protein complexes and, to a lesser extent, in GCS(l)EpRE-protein complexes. The Nrf2-associated expression of GCS promoter/reporter transgenes was inhibited by overexpression of MafG. Inhibition of protein synthesis by cycloheximide partially decreased inducibility by PDTC or beta-NF and resulted in significant increases in GCS mRNA at late time points, when GCS mRNA levels are normally declining. We hypothesize that, in response to beta-NF and PDTC, the GCS subunit genes are transcriptionally up-regulated by Nrf2-basic leucine zipper complexes, containing either JunD or small Maf protein, depending on the particular GCS EpRE target sequence and the inducer. Following maximal induction, down-regulation of the two genes is mediated via a protein synthesis-dependent mechanism.
The importance of dopaminergic transmission for normal behaviour has been evident since the initial characterization of the organization and functioning of the dopamine (DA) pathways, as well as the subsequent discovery and mapping of the DA receptor systems, comprising the D1-like receptors (i.e. D1, D4 and D5 receptor subtypes) and the D2/3-like receptors. The search for functional correlates of DA function has been given great impetus by its undoubted involvement in Parkinson’s disease, in the mediation of reinforcing effects of drugs of abuse such as the amphetamine-like psychomotor stimulants and in the anti-psychotic effects of neuroleptic drugs. The purpose of this chapter is to build upon the syntheses provided by several previous reviews and to reach conclusions about the nature of the contribution of DA neurotransmission to behaviour, with particular emphasis on its possible role in cognition.
Formation of nitric oxide (NO), by astrocytes, has been suggested to contribute, via impairment of mitochondrial function, to the neurodegnerative process. Thus co‐culture of neuronal cells with NO–generating astrocytes leads to a loss of mitochondrial function, as reflected by diminished activities of complexes IV and II+III. However, such damage may in the first instance be limited due to upregulation of neuronal glutathione metabolism as a result of metabolic trafficking of glutathione from the astrocyte to neurone. Furthermore, exposure of astrocytes to NO leads to increased glutathione metabolism resulting in the preservation of glutathione precursors for neuronal utilization. Failure of glutathione trafficking could render neuronal cells particularly susceptible to NO, leading to cell death. In addition, depletion with time of the nitric oxide synthase cofactor, tetrahydrobiopterin, may result in the astrocytic generation of more potent oxidizing species, which could contribute to the neurodegenerative process.
The effects of dopamine and L-DOPA on survival were examined in differentiated PC12 cells. Addition of dopamine to the culture medium at 3–30 μM prevented cell death induced by depletion of serum and nerve growth factor (NGF). At 100 μM, dopamine induced cell death. The cell-protective effect of dopamine was not affected by nomifensine, an inhibitor of dopamine uptake, or pargyline, an inhibitor of monoamine oxidase, suggesting that dopamine is working outside the cell. The cell-protective effect of dopamine was blunted by SCH-23390, a D1 antagonist, but not sulpiride, a D2 antagonist, indicating that the cell protective effect of dopamine is mediated by D1 receptors in PC12 cells. L-DOPA also protected PC12 cells from cell death induced by depletion of serum and NGF at low concentrations and showed toxicity at high concentration. The effect of L-DOPA was unchanged after inhibition of conversion of L-DOPA to dopamine by m-hydroxybenzylhydrazine (NSD-1015), an inhibitor of DOPA decarboxylase, suggesting that L-DOPA itself is working for cell protection. Intracellular Ca2+ concentration and mitogen-activated protein (MAP) kinase activity were increased by both dopamine and L-DOPA. The effects of dopamine and L-DOPA on cell survival were blunted by nicardipine, a Ca2+ channel blocker, and PD-98059, an inhibitor of MAP kinase kinase (MEK). These results taken together raised the possibility that dopamine and L-DOPA protect PC12 cells from cell death at low concentrations by activating MAP kinase activity via elevation of intracellular Ca2+ concentration. J. Neurosci. Res. 62:112–119, 2000. © 2000 Wiley-Liss, Inc.
Incubation with l-DOPA induced a rise in GSH level in cultures of fetal rat mesencephalon, mouse neuroblastoma (Neuro-2A), human neuroblastoma (SK-N-MC), pig kidney epithelial cells (LLC-PK1), and glia from newborn rat brain, but not C6 glioma cells or neuronal cultures (no glia) from the mesencephalon. The pure neuronal cultures were destroyed by incubation with l-DOPA; added ascorbic acid or superoxide dismutase protected the cells. Washout of l-DOPA after 48 h amplified the rise in GSH content in mixed cultures (neurons plus glia). Examination of structure-activity relationships for elevating GSH levels in responsive cell types revealed that autooxidizable compounds (α-methyl-DOPA, dopamine, apomorphine, catechol, and hydroquinone) behaved similarly to l-DOPA, whereas structural analogues that cannot undergo autooxidation (3-O-methyl-DOPA, tyrosine, 2,4-dihydroxyphenylalanine, and resorcinol) failed to elevate GSH levels. Therefore, up-regulation of GSH appears to be a response to a mild oxidative stress. When mixed mesencephalic cultures were exposed to a strong oxidant stress by incubation with tert-butyl hydroperoxide, a loss in viability was seen. Cultures pretreated with l-DOPA or hydroquinone were protected from loss of viability. However, when cultures were pretreated with both l-DOPA and ascorbate, which prevents the rise in GSH level, protection was lost, in accord with the failure to up-regulate GSH. These results show that the up-regulation of cellular GSH evoked by autooxidizable agents is associated with significant protection of cells. Glia play an essential role in the response of mesencephalic cell cultures. An ability to up-regulate GSH may serve a protective role in vivo.
The levels of the antioxidants, glutathione and ascorbate were measured in primary cultures of murine astrocytes and neurons. The concentration of glutathione (reduced, GSH + oxidized, GSSG) was high in cultured, differentiated (i.e. treated with dBcAMP) and undifferentiated (i.e. untreated) astrocytes:≈25 (n = 2)and16.0 ± 5.0 (n = 7)nmol/mg protein, respectively. In contrast, glutathione levels in neurons were low:≦1.0 (n = 7)nmol/mg protein. Ascorbate could not be detected (<2nmol/mg protein) in either cell type. The apparent lack of defense mechanisms against oxidative stress may in part account for the ‘fragility’ of neurons in culture. The physiological implications of glutathione compartmentation in brain are discussed.
The monoamine neurotransmitter disorders consist of a rapidly expanding heterogeneous group of neurological syndromes characterised by primary and secondary defects in the biosynthesis degradation, or transport of dopamine, norepinephrine, epinephrine, and serotonin. Disease onset can occur any time from infancy onwards. Clinical presentation depends on the pattern and severity of neurotransmitter abnormalities, and is predominated by neurological features (encephalopathy, epilepsy, and pyramidal and extrapyramidal motor disorders) that are primarily attributed to deficiency of cerebral dopamine, serotonin, or both. Many neurotransmitter disorders mimic the phenotype of other neurological disorders (eg, cerebral palsy, hypoxic ischaemic encephalopathy, paroxysmal disorders, inherited metabolic diseases, and genetic dystonic or parkinsonian syndromes) and are, therefore, frequently misdiagnosed. Early clinical suspicion and appropriate investigations, including analysis of neurotransmitters in CSF, are essential for accurate clinical diagnosis. Treatment strategies focus on the correction of monoamine deficiency by replacement of monoamine precursors, the use of monoamine analogues, inhibition of monoamine degradation, and addition of enzyme cofactors to promote monoamine production.
Mitochondrial encephalomyopathies, arising from deficiencies of the electron transport chain (ETC) give rise to a wide clinical spectrum of presentation and are often progressive in nature. The aetiology of mitochondrial encephalomyopathies have yet to be fully elucidated, however, a successive loss of ETC function may contribute to the progressive nature of these disorders. The possibility arises that as a consequence of a primary impairment of ETC activity, secondary damage to the ETC may occur. In order to investigate this hypothesis, we established a model of cytochrome oxidase (Complex IV) deficiency in cultured human astrocytoma 1321N cells. Potassium cyanide (KCN, 1mM) resulted in a sustained 50% (p<0.01) loss of complex IV. At 24h activities of the other ETC complexes were unaffected. However, at 72h significant loss of succinate-cytochrome c reductase (complex II-III) activity expressed as a ratio to the mitochondrial marker, citrate synthase was observed. (KCN treated; 0.065+/-0.011 vs controls; 0.118+/-0.017 mean+/-SEM, n=8, p<0.05). These results provide a possible mechanism for the progressive nature of ETC defects and why in some patients multiple patterns of ETC deficiencies can be demonstrated.
The transcription factor Nrf2 controls inducible expression of multiple antioxidant/detoxification genes. We previously found that Nrf2-/- mice have increased sensitivity to in vivo mitochondrial stress and ischemia. Although Nrf2 regulated these forms of neuronal toxicity, it was unclear which injury-triggered signal(s) led to Nrf2 activation in vivo. In this study, we use primary cultures to test the hypothesis that excessive dopamine release can act as an endogenous Nrf2-inducing signal. We cultured two cell types that show increased Nrf2 activity during ischemia in vivo, astrocytes and meningeal cells. Cultures were infected with an adenovirus reporter of Nrf2 transcriptional activity. Dopamine-induced Nrf2 activity in both cell types by generating oxidative stressors, H2O2 and dopamine-quinones. Nrf2 activation in meningeal cells was significantly higher than astrocytes. The effect of dopamine was blocked by antioxidants, and by over-expression of either dominant-negative Nrf2 or Keap1. Nrf2 induction was specific to oxidative stress caused by catecholaminergic neurotransmitters as epinephrine also induced Nrf2, but the monoamine serotonin had no significant effect. These in vitro results suggest Nrf2 activity in astrocytes and meningeal cells link the neurotoxic actions of dopamine to neuroprotective pathways that may potentially modulate ischemic injury and neurodegeneration.
To describe the current treatment; clinical, biochemical, and molecular findings; and clinical follow-up of patients with aromatic l-amino acid decarboxylase (AADC) deficiency.
Clinical and biochemical data of 78 patients with AADC deficiency were tabulated in a database of pediatric neurotransmitter disorders (JAKE). A total of 46 patients have been previously reported; 32 patients are described for the first time.
In 96% of AADC-deficient patients, symptoms (hypotonia 95%, oculogyric crises 86%, and developmental retardation 63%) became clinically evident during infancy or childhood. Laboratory diagnosis is based on typical CSF markers (low homovanillic acid, 5-hydroxyindoleacidic acid, and 3-methoxy-4-hydroxyphenolglycole, and elevated 3-O-methyl-l-dopa, l-dopa, and 5-hydroxytryptophan), absent plasma AADC activity, or elevated urinary vanillactic acid. A total of 24 mutations in the DDC gene were detected in 49 patients (8 reported for the first time: p.L38P, p.Y79C, p.A110Q, p.G123R, p.I42fs, c.876G>A, p.R412W, p.I433fs) with IVS6+ 4A>T being the most common one (allele frequency 45%).
Based on clinical symptoms, CSF neurotransmitters profile is highly indicative for the diagnosis of aromatic l-amino acid decarboxylase deficiency. Treatment options are limited, in many cases not beneficial, and prognosis is uncertain. Only 15 patients with a relatively mild form clearly improved on a combined therapy with pyridoxine (B6)/pyridoxal phosphate, dopamine agonists, and monoamine oxidase B inhibitors.
J. Neurochem. (2010) 114, 87–96.
Pyridoxal 5′-phosphate, the active form of vitamin B6, is an essential cofactor for multiple enzymes, including aromatic l-amino acid decarboxylase that catalyses the final stage in the production of the neurotransmitters dopamine and serotonin. In two patients with inherited disorders of vitamin B6 metabolism, we observed reductions in plasma aromatic l-amino acid decarboxylase activity. In one patient, this change was related to an increase in Km for pyridoxal 5′-phosphate. Furthermore, pyridoxal 5′-phosphate-deficient human SH-SY5Y neuroblastoma cells were found to exhibit reduced levels of aromatic l-amino acid decarboxylase activity and protein but with no alteration in expression. Further reductions in activity and protein were observed with the addition of the vitamin B6 antagonist 4-deoxypyridoxine, which also reduced aromatic l-amino acid decarboxylase mRNA levels. Neither pyridoxal 5′-phosphate deficiency nor the addition of 4-deoxypyridoxine affected aromatic l-amino acid decarboxylase stability over 8 h with protein synthesis inhibited. Increasing extracellular availability of pyridoxal 5′-phosphate was not found to have any significant effect on intracellular pyridoxal 5′-phosphate concentrations or on aromatic l-amino acid decarboxylase. These findings suggest that maintaining adequate pyridoxal 5′-phosphate availability may be important for optimal treatment of aromatic l-amino acid decarboxylase deficiency and l-dopa-responsive conditions.
Transcription factor NF-E2 p45-related factor 2 (Nrf2) mediates adaptation to oxidants and electrophiles through up-regulating genes that contain antioxidant response elements (AREs) in their promoters. Using the stably transfected human AREc32 reporter cell line, we found that copper and other transition metals enhanced induction of ARE-driven luciferase by 2-tert-butyl-1,4-hydroquinone (tBHQ) as a result of increased oxidation to 2-tert-butyl-1,4-benzoquinone (tBQ). Following exposure to tBHQ for 30 min, ARE-luciferase activity measured after 24 hr was dependent on the presence of Cu(2+). In contrast, tBQ-induced activity was Cu(2+)-independent. The metal-catalyzed oxidation of tBHQ to tBQ occured rapidly and stoichiometrically. Compounds that share para- or ortho-hydroquinone structures, such as catechol estrogens, dopamine, and l-DOPA, also induced ARE-driven luciferase in a Cu(2+)-dependent manner. Thus, the oxidation of para- and ortho-hydroquinones to quinones represents the rate-limiting step in the activation of Nrf2.
Nrf2:INrf2 (Keap1) are cellular sensors of chemical- and radiation-induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor, INrf2 which functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2 cysteine 151 and/or protein kinase C phosphorylation of Nrf2 serine 40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds the antioxidant response element, which leads to coordinated activation of gene expression. It takes less than 15 min from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls the switching off of Nrf2 activation of gene expression. GSK3beta phosphorylates Fyn at an unknown threonine residue(s), leading to the nuclear localization of Fyn. Fyn phosphorylates Nrf2 tyrosine 568, resulting in the nuclear export of Nrf2, binding with INrf2, and degradation of Nrf2. The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis, and promotes cell survival.
Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting over a million people in the United States alone, and is characterized by rigidity, bradykinesia, resting tremor, and postural instability. Its main neuropathological feature is the loss of dopaminergic neurons of the substantia nigra pars compacta. However, the pathogenesis of this loss is not understood fully. One of the earliest biochemical changes seen in PD is a reduction in the levels of total glutathione, a key cellular antioxidant. Traditionally, it has been thought that this decrease in GSH levels is the consequence of increased oxidative stress, a process heavily implicated in PD pathogenesis. However, emerging evidence suggests that GSH depletion may itself play an active role in PD pathogenesis. This review aims to explore the contribution of GSH depletion to PD pathogenesis.
Incubation of cultured rat glomerular mesangial cells with dopamine caused an increase in cyclic AMP formation in a concentration-dependent manner (Ka apparent 2.2 microM). The selective dopamine D1 receptor agonists, fenoldopam, SKF 38393 and (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN) also produced concentration-dependent increases in cyclic AMP with mean Ka apparent values of 0.04 microM, 0.02 microM and 1.02 microM, respectively. Although fenoldopam and SKF 38393 were more potent than dopamine, they were partial agonists with efficacies, relative to dopamine, of approximately 60 and 35%, respectively. The dopamine analogue, 6,7-ADTN, in contrast, behaved as a full agonist. Dopamine-stimulated cAMP formation was inhibited in a concentration-dependent manner by the D1-selective antagonist, SCH 23390, with a Ki of 0.06 nM. In contrast, the D2-selective antagonist, domperidone, was four orders of magnitude less potent than SCH 23390, having a Ki of 2072 nM. In addition, SCH 23388, the stereoisomer of SCH 23390, was observed to be two orders of magnitude less potent than SCH 23390, indicating the stereoselective nature of the receptor. The potency series for the selective agonists and antagonists is the same as that described, using identical experimental conditions, for the D1 receptor expressed by a cell line of central origin confirming that the peripheral DA1 and the central D1 dopamine receptor are pharmacologically similar.
The regional distributions of iron, copper, zinc, magnesium, and calcium in parkinsonian brains were compared with those of matched controls. In mild Parkinson's disease (PD), there were no significant differences in the content of total iron between the two groups, whereas there was a significant increase in total iron and iron (III) in substantia nigra of severely affected patients. Although marked regional distributions of iron, magnesium, and calcium were present, there were no changes in magnesium, calcium, and copper in various brain areas of PD. The most notable finding was a shift in the iron (II)/iron (III) ratio in favor of iron (III) in substantia nigra and a significant increase in the iron (III)-binding, protein, ferritin. A significantly lower glutathione content was present in pooled samples of putamen, globus pallidus, substantia nigra, nucleus basalis of Meynert, amygdaloid nucleus, and frontal cortex of PD brains with severe damage to substantia nigra, whereas no significant changes were observed in clinicopathologically mild forms of PD. In all these regions, except the amygdaloid nucleus, ascorbic acid was not decreased. Reduced glutathione and the shift of the iron (II)/iron (III) ratio in favor of iron (III) suggest that these changes might contribute to pathophysiological processes underlying PD.
Glutathione (GSH) is a ubiquitous cellular sulfhydryl compound with a variety of essential functions. A histochemical method that was developed by others for the localization of GSH in tissue sections was used to study the localization of GSH in rodent and primate brain. Sections of freshly frozen tissue were stained for 4 min with Mercury orange dissolved in toluene, and viewed by fluorescence microscopy for the product of the reaction with soluble sulfhydryl compounds. Soluble sulfhydryl compounds are comprised almost exclusively of GSH. Although the brain exhibits strong staining characteristics, reflecting the millimolar levels of GSH that are detected by chemical assay, very little stain is seen in neuronal somata. Pretreatment of animals with diethyl maleate resulted in depletion of GSH from brain (measured by high performance liquid chromatography), as well as decreased Mercury orange staining. The staining pattern observed in the brain may indicate that GSH is primarily localized to non-neuronal elements, such as glia, and/or in axons and nerve terminals.
Fourteen human neuroblastoma cell lines were studied for expression and regulation of neurotransmitter-synthesizing enzymes. All cell lines contained activities of adrenergic and/or cholinergic neurons and 13 expressed activities for both. None contained enzymes for serotonergic or GABAergic neurons. Enzyme activity was characteristic for a given cell line. Enzyme activity in cell lines was sensitive to growth phase, culture medium, and concentration of fetal bovine serum.
The Ca(2+)-independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 microgram/ml) plus interferon-gamma (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH-ubiquinone-1 reductase (complex I) and succinate-cytochrome c reductase (complex II-III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate-cytochrome c reductase activity, but NADH-ubiquinone-1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The lipopolysaccharide/interferon-gamma treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide-mediated neurotoxicity.
The effect of the neurotoxic nitric oxide derivative, the peroxynitrite anion (ONOO-), on the activity of the mitochondrial respiratory chain complexes in cultured neurones and astrocytes was studied. A single exposure of the neurones to ONOO- (initial concentrations of 0.01-2.0 mM) caused, after a subsequent 24-h incubation, a dose-dependent decrease in succinate-cytochrome c reductase (60% at 0.5 mM) and in cytochrome c oxidase (52% at 0.5 mM) activities. NADH-ubiquinone-1 reductase was unaffected. In astrocytes, the activity of the mitochondrial complexes was not affected up to 2 mM ONOO-. Citrate synthase was unaffected in both cell types under all conditions studied. However, lactate dehydrogenase activity released to the culture medium was increased by ONOO- in a dose-dependent manner (40% at 0.5 mM ONOO-) from the neurones but not from the astrocytes. Neuronal glutathione concentration decreased by 39% at 0.1 mM ONOO-, but astrocytic glutathione was not affected up to 2 mM ONOO-. In isolated brain mitochondria, only succinate-cytochrome c reductase activity was affected (22% decrease at 1 mM ONOO-). We conclude that the acute exposure of ONOO- selectively damages neurones, whereas astrocytes remain unaffected. Intracellular glutathione appears to be an important factor for ameliorating ONOO(-)-mediated mitochondrial damage. This study supports the hypothesis that the neurotoxicity of nitric oxide is mediated through mitochondrial dysfunction.
The effect of depletion of reduced glutathione (GSH) on brain mitochondrial function and N-acetyl aspartate concentration has been investigated. Using pre-weanling rats, GSH was depleted by L-buthionine sulfoximine administration for up to 10 days. In both whole brain homogenates and purified mitochondrial preparations complex IV (cytochrome c oxidase) activity was decreased, by up to 27%, as a result of this treatment. In addition, after 10 days of GSH depletion, citrate synthase activity was significantly reduced, by 18%, in the purified mitochondrial preparations, but not in whole brain homogenates, suggesting increased leakiness of the mitochondrial membrane. The whole brain N-acetyl aspartate concentration was also significantly depleted at this time point, by 11%. It is concluded that brain GSH is important for the maintenance of optimum mitochondrial function and that prolonged depletion leads also to loss of neuronal integrity. The relevance of these findings to Parkinson's disease and the inborn errors of glutathione metabolism are also discussed.
Mesenteric artery vascular smooth muscle cells derived from male Wistar rats and grown in culture were prelabelled with [ ³ H]‐adenine and exposed to a range of dopamine receptor agonists and antagonists. Resultant [ ³ H]‐cyclic AMP formation was determined and concentration‐effect curves constructed, in the presence of propranolol (10 ⁻⁶ m ) and the phosphodiesterase inhibitor IBMX (5× 10 ⁻⁴ m ).
K a apparent values for D 1 /DA 1 dopamine receptor agonists SKF 38393, fenoldopam, 6,7‐ADTN, and dopamine were 0.06, 0.59, 4.06 and 5.77 × 10 ⁻⁶ m respectively. Although fenoldopam and SKF 38393 were more potent than dopamine, they were partial agonists with efficacies, relative to dopamine of approximately 48% and 24% respectively. 6,7‐ADTN, in contrast, behaved as a full agonist.
Dopamine‐stimulated cyclic AMP formation was inhibited in a concentration‐dependent manner by the D 1 /DA 1 dopamine receptor selective antagonists, SCH 23390 and cis ‐flupenthixol ( K i values 0.53 and 36.1 × 10 ⁻¹ m respectively). In contrast, the D 2 /DA 2 dopamine receptor selective antagonists, domperidone and (−)‐sulpiride, were less potent ( K i values 2.06 and 5.82 × 10 ⁻⁶ m respectively). Furthermore, the stereoisomers of SCH 23390 and cis ‐flupenthixol, SCH 23388 and trans‐flupenthixol, were at least two orders of magnitude less potent ( K i values 0.14 and 13.2 × 10 ⁻⁶ m respectively) indicating the stereoselective nature of this receptor.
Our results indicate that rat mesenteric artery vascular smooth muscle cells in culture express a dopamine receptor coupled to cyclic AMP formation, which has the pharmacological profile, characteristic of the D 1 dopamine receptor subfamily.
GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included gamma-glutamylcysteine synthetase, GSH synthetase, gamma-glutamyl cyclotransferase, gamma-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. gamma-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)
The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 microM) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [3H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions. Ascorbate (200 microM), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [3H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, L-DOPA prevented cell death by L-BSO.
Levodopa, a dopamine (DA) precursor administered to patients with Parkinson's disease (PD), produces at 25-200 x 10(-6) M concentrations a dose-dependent reduction of 3H-DA uptake in foetal rat midbrain cultures. Also, a decrease in the number of viable cells and tyrosine hydroxylase (TH) positive neurones, plus disruption of the overall neuritic network are observed concurrently with an elevation of quinone levels in the culture medium. Ascorbic acid (AA), which abolished the quinone overproduction, partially prevented these effects. Though levodopa neurotoxicity in vivo is as yet unproven, AA may reduce vulnerability of endogenous or grafted DA neurones in patients with PD.
L-DOPA kills dopamine neurones in culture but is the most effective drug for the treatment of Parkinson's disease, where it exhibits no clear toxicity. While glial cells surround and protect neurones in vivo, neurones are usually cultured in vitro in the absence of glia. We treated fetal midbrain rat neurones with L-DOPA, mesencephalic glia conditioned medium (CM) and L-DOPA + CM. L-DOPA reduced the number of tyrosine hydroxylase-positive (TH+) cells and [3H]DA uptake, and increased quinone levels. L-DOPA + CM restored [3H]DA uptake and quinone levels to normal, and increased the number of TH+ cells and terminals to 170% of control. CM greatly increased the number of TH+ cells and [3H]DA uptake. Mesencephalic glia therefore produced soluble factors which are neurotrophic for dopamine neurones, and which protect these neurones from the toxic effects of L-DOPA.
Loss of the intracellular antioxidant glutathione (GSH) from the substantia nigra is considered to be an early event in the pathogenesis of Parkinson's disease (PD). While the cause of the loss is unclear, an imbalance in the enzymes associated with the synthesis, utilisation, degradation and translocation of GSH has been implicated. The enzyme glutathione reductase is also important in GSH homeostasis: it regenerates GSH from the oxidised from (GSSG). However, to date the activity and regulation of glutathione reductase in conditions such as PD have not been explored. In view of this we have measured the effects of GSH depletion on glutathione reductase activity of the rat brain. Other glutathione related enzymes were also measured. Using pre-weanling rats, brain GSH was depleted by up to 60% by subcutaneous administration of L-buthionine sulfoximine. The only enzyme affected by GSH depletion was glutathione reductase; its activity being reduced by approximately 40%. As GSH inactivates a number of oxidising species including peroxynitrite (ONOO-), we additionally investigated the susceptibility of glutathione reductase to ONOO- in vitro, using purified enzyme. ONOO- decreased glutathione reductase activity in a concentration dependent manner with an apparent 50% inhibition occurring at an initial concentration of 0.09 mM. These data suggest that GSH is important in the maintenance glutathione reductase activity. This may arise in part from its ability to inactivate oxidising agents such as ONOO-.
The characteristics and kinetics of GSH efflux from the monolayer culture of rat astrocytes were investigated. GSH efflux was dependent on temperature, with a Q10 value of 2.0 between 37 and 25 degrees C. The GSH efflux rate showed a hyperbolic dependency on the intracellular GSH concentration. The data were fitted well to the Michaelis-Menten model, giving the following kinetic parameter values: Km = 127 nmol/mg of protein; Vmax = 0.39 nmol/min/mg of protein. p-Chloromercuribenzenesulfonic acid, a thiol-reactive agent impermeable to the cell membrane, lowered the GSH efflux rate by 25% without affecting the intracellular GSH content. These results suggest that a carrier is involved in the efflux of GSH. The GSH content of cultured astrocytes showed a marked increase when the cells were exposed to insults, such as sodium arsenite, cadmium chloride, and glucose/glucose oxidase that lead to the generation of hydrogen peroxide. The increase in GSH content was attributed to the induction of the cystine transport activity by the agents. Although the intracellular GSH concentration and GSH efflux were increased, the kinetics of GSH efflux were not affected by those agents that imposed the oxidative stress. Because the Km value is very large, it is suggested that astrocytes release GSH depending on their GSH concentration in a wide range.
The effect of glutathione depletion, in vivo, on rat brain nitric oxide synthase activity has been investigated and compared to the effect observed in vitro with cultured neurones. Using L-buthionine sulfoximine rat brain glutathione was depleted by 62%. This loss of glutathione was accompanied by a significant increase in brain nitric oxide synthase activity by up to 55%. Depletion of glutathione in cultured neurones, by approximately 90%, led to a significant 67% increase in nitric oxide synthase activity, as judged by nitrite formation, and cell death. It is concluded that depletion of neuronal glutathione results in increased nitric oxide synthase activity. These findings may have implications for our understanding of the pathogenesis of neurodegenerative disorders in which loss of brain glutathione is considered to be an early event.
In this study we have investigated the mechanisms leading to mitochondrial damage in cultured neurons following sustained exposure to nitric oxide. Thus, the effects upon neuronal mitochondrial respiratory chain complex activity and reduced glutathione concentration following exposure to either the nitric oxide donor, S-nitroso-N-acetylpenicillamine, or to nitric oxide releasing astrocytes were assessed. Incubation with S-nitroso-N-acetylpenicillamine (1 mM) for 24 h decreased neuronal glutathione concentration by 57%, and this effect was accompanied by a marked decrease of complex I (43%), complex II-III (63%), and complex IV (41%) activities. Incubation of neurons with the glutathione synthesis inhibitor, L-buthionine-[S,R]-sulfoximine caused a major depletion of neuronal glutathione (93%), an effect that was accompanied by a marked loss of complex II-III (60%) and complex IV (41%) activities, although complex I activity was only mildly decreased (34%). In an attempt to approach a more physiological situation, we studied the effects upon glutathione status and mitochondrial respiratory chain activity of neurons incubated in coculture with nitric oxide releasing astrocytes. Astrocytes were activated by incubation with lipopolysaccharide/interferon-gamma for 18 h, thereby inducing nitric oxide synthase and, hence, a continuous release of nitric oxide. Coincubation for 24 h of activated astrocytes with neurons caused a limited loss of complex IV activity and had no effect on the activities of complexes I or II-III. However, neurons exposed to astrocytes had a 1.7-fold fold increase in glutathione concentration compared to neurons cultured alone. Under these coculture conditions, the neuronal ATP concentration was modestly reduced (14%). This loss of ATP was prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. These results suggest that the neuronal mitochondrial respiratory chain is damaged by sustained exposure to nitric oxide and that reduced glutathione may be an important defence against such damage.
Enhanced oxidative stress has been suggested to be involved in the degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease. The high turnover rate of dopamine and/or unsequestered dopamine may cause an increase of formation of hydrogen peroxide via either oxidative deamination of dopamine by monoamine oxidase or autoxidation. Hydrogen peroxide would be converted to more toxic hydroxyl free radicals. L-beta-3,4-Dihydroxyphenylalanine hydrochloride (L-DOPA), the most useful drug in the symptomatic treatment of Parkinson's disease, has been considered to possess deteriorating degenerative side-effects. The catecholaminergic neuroblastoma SH-SY5Y cells were chosen to investigate the cytotoxic effect of dopamine and L-DOPA. Both dopamine and L-DOPA were found to be cytotoxic towards SH-SY5Y cells. Such toxic effects were accompanied by an increase of oxidative stress in the cell cultures and could be reversed effectively by catalase and to a lesser extent by superoxide dismutase. The non-enzymatic antioxidants L-ascorbic acid, glutathione, N-acetyl-L-cysteine, but not (+)-alpha-tocopherol, also completely protected SH-SY5Y cells against the cytotoxic effects induced by dopamine and L-DOPA. Antioxidative factors, namely free radical scavengers (including N-tert-butyl-alpha-phenylnitrone, salicylic acid, and D-mannitol) and a strong iron chelator, deferoxamine, however, did not protect the SH-SY5Y cells against dopamine and L-DOPA. The generation of reactive oxygen species and the resulting enhanced oxidative stress was clearly involved in the dopamine- and L-DOPA-induced cytotoxic effects. Hydrogen peroxide played the most important role related to cytotoxicity of dopamine and L-DOPA.
The release of glutathione from astroglial cells was investigated using astroglia-rich primary cultures prepared from the brains of newborn rats. These cells release glutathione after onset of an incubation in a glucose-containing minimal medium. The amount of extracellular glutathione increased with the time of incubation, although the accumulation slowed down gradually. An elevated rate of increase of the glutathione concentration in the incubation medium was found if the astroglial ectoenzyme gamma-glutamyl transpeptidase was inhibited by acivicin. The activity of gamma-glutamyl transpeptidase in astroglia-rich primary cultures, which was found to be 1.9 +/- 0.3 nmol/(min x mg protein), was markedly reduced if the cells had been incubated in the presence of acivicin. After 2 h of incubation with acivicin half-maximal and maximal inhibition of gamma-glutamyl transpeptidase activity was found at concentrations of about 5 microM and 50 microM, respectively. In the presence of acivicin at a concentration above 10 microM the glutathione content found released from astroglial cells apparently increased almost proportional to time for up to 10 h. Under these conditions the average rate of release was 2.1 +/- 0.3 nmol/(h x mg protein) yielding after a 10 h incubation an extracellular glutathione content three times that of the medium of cells incubated without inhibitor. Half-maximal and maximal effects on the level of extracellular glutathione were found at 4 microM and 50 microM acivicin, respectively. After a 10 h incubation with acivicin the intracellular content of glutathione was reduced to 75% of the level of untreated astroglial cultures. These results suggest that glutathione released from astroglial cells can serve as substrate for the ectoenzyme gamma-glutamyl transpeptidase of these cells.
Mesencephalic glia produce soluble factors that protect dopamine neurons from L-DOPA toxicity. The chemical composition of these soluble factors is unknown. We investigated the protective effect against L-DOPA neurotoxicity in midbrain dopamine neurons of fractions of different molecular size of glia conditioned medium and candidate neuroprotective agents produced by glia including neurotrophic factors and antioxidants. Protective effects were evaluated according to the number of tyrosine hydroxylase immunoreactive cells, high affinity dopamine uptake and levels of quinones. Both fractions of glia conditioned medium, smaller and larger than 10kD, protected against L-DOPA, but the fraction of smaller molecular size, that contains small free radical scanvenger molecules, was more effective than the fraction of larger molecular size, that contains large neurotrophic peptides. Among the neurotrophic factors GDNF and BDNF totally prevented L-DOPA neurotoxicity, while NGF and bFGF were less effective. However, only NGF significantly reduced the elevation of quinones induced by L-DOPA. Ascorbic acid, at the concentration found in glia conditioned medium, provided partial protective effect against L-DOPA toxicity. Glutathione, had neurotrophic effects on untreated midbrain dopamine neurons and prevented the effect of L-DOPA. In conclusion, the protective effect against L-DOPA neurotoxicity by glia conditioned medium is mediated by several compounds including neurotrophic factors and small antioxidants.
L-DOPA is toxic to catecholamine neurons in culture, but the toxicity is reduced by exposure to astrocytes. We tested the effect of L-DOPA on dopamine neurons using postnatal ventral midbrain neuron/cortical astrocyte cocultures in serum-free, glia-conditioned medium. L-DOPA (50 microM) protected against dopamine neuronal cell death and increased the number and branching of dopamine processes. In contrast to embryonically derived glia-free cultures, where L-DOPA is toxic, postnatal midbrain cultures did not show toxicity at 200 microM L-DOPA. The stereoisomer D-DOPA (50-400 microM) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 microM) did not block the neurotrophic effect. These data suggest that the neurotrophic effect of L-DOPA is stereospecific but independent of the production of dopamine. However, L-DOPA increased the level of glutathione. Inhibition of glutathione peroxidase by L-buthionine sulfoximine (3 microM for 24 h) blocked the neurotrophic action of L-DOPA. N-Acetyl-L-cysteine (250 microM for 48 h), which promotes glutathione synthesis, had a neurotrophic effect similar to that of L-DOPA. These data suggest that the neurotrophic effect of L-DOPA may be mediated, at least in part, by elevation of glutathione content.
The diverse physiological actions of dopamine are mediated by at least five distinct G protein-coupled receptor subtypes. Two D1-like receptor subtypes (D1 and D5) couple to the G protein Gs and activate adenylyl cyclase. The other receptor subtypes belong to the D2-like subfamily (D2, D3, and D4) and are prototypic of G protein-coupled receptors that inhibit adenylyl cyclase and activate K+ channels. The genes for the D1 and D5 receptors are intronless, but pseudogenes of the D5 exist. The D2 and D3 receptors vary in certain tissues and species as a result of alternative splicing, and the human D4 receptor gene exhibits extensive polymorphic variation. In the central nervous system, dopamine receptors are widely expressed because they are involved in the control of locomotion, cognition, emotion, and affect as well as neuroendocrine secretion. In the periphery, dopamine receptors are present more prominently in kidney, vasculature, and pituitary, where they affect mainly sodium homeostasis, vascular tone, and hormone secretion. Numerous genetic linkage analysis studies have failed so far to reveal unequivocal evidence for the involvement of one of these receptors in the etiology of various central nervous system disorders. However, targeted deletion of several of these dopamine receptor genes in mice should provide valuable information about their physiological functions.
The cause of neuronal cell death in Parkinson's disease is unknown but there is accumulating evidence suggesting that oxidative stress may be involved in this process. Current evidence shows that in the substantia nigra there is altered iron metabolism, decreased levels of reduced glutathione and an impairment of mitochondrial complex I activity. However, these changes seem to be unique to the substantia nigra and have not been found in other areas of the brain known to be altered in Parkinson's disease, such as substantia innominata. In addition they do not appear to be related to the presence of Lewy bodies, as other areas of the brain containing Lewy bodies do not show evidence of either oxidative stress or mitochondrial dysfunction. Oxidative stress has now been demonstrated in Alzheimer's disease and its presence appears to be correlated with regions of marked pathological changes.
The cause of neuronal loss in patients with idiopathic Parkinson's disease is unknown. Oxidative stress and complex I deficiency have both been identified in the substantia nigra in Parkinson's disease but their place in the sequence of events resulting in dopaminergic cell death is uncertain. We have analysed respiratory chain activity, iron and reduced glutathione concentrations in Parkinson's disease substantia innominata and in the cingulate cortex of patients with Parkinson's disease, Alzheimer's disease and dementia with Lewy bodies to investigate their association with neuronal death and Lewy body formation. No abnormalities of mitochondrial function, iron or reduced glutathione levels were identified in Parkinson's disease substantia innominata or cingulate cortex. Mitochondrial function also appeared to be unchanged in cingulate cortex from patients with Alzheimer's disease and from patients with dementia with Lewy bodies, however, iron concentrations were mildly increased in both, and reduced glutathione decreased only in Alzheimer's disease. These results confirm the anatomic specificity of the complex I deficiency and decreased levels of reduced glutathione within the Parkinson's disease brain and suggest that these parameters are not associated with cholinergic cell loss in Parkinson's disease nor with Lewy body formation in this or other diseases. We propose that our data support a 'two-hit' hypothesis for the cause of neuronal death in Parkinson's disease.
The effects of dopamine and L-DOPA on survival were examined in differentiated PC12 cells. Addition of dopamine to the culture medium at 3-30 microM prevented cell death induced by depletion of serum and nerve growth factor (NGF). At 100 microM, dopamine induced cell death. The cell-protective effect of dopamine was not affected by nomifensine, an inhibitor of dopamine uptake, or pargyline, an inhibitor of monoamine oxidase, suggesting that dopamine is working outside the cell. The cell-protective effect of dopamine was blunted by SCH-23390, a D(1) antagonist, but not sulpiride, a D(2) antagonist, indicating that the cell protective effect of dopamine is mediated by D(1) receptors in PC12 cells. L-DOPA also protected PC12 cells from cell death induced by depletion of serum and NGF at low concentrations and showed toxicity at high concentration. The effect of L-DOPA was unchanged after inhibition of conversion of L-DOPA to dopamine by m-hydroxybenzylhydrazine (NSD-1015), an inhibitor of DOPA decarboxylase, suggesting that L-DOPA itself is working for cell protection. Intracellular Ca(2+) concentration and mitogen-activated protein (MAP) kinase activity were increased by both dopamine and L-DOPA. The effects of dopamine and L-DOPA on cell survival were blunted by nicardipine, a Ca(2+) channel blocker, and PD-98059, an inhibitor of MAP kinase kinase (MEK). These results taken together raised the possibility that dopamine and L-DOPA protect PC12 cells from cell death at low concentrations by activating MAP kinase activity via elevation of intracellular Ca(2+) concentration.
Neurons in culture rely on the supply of exogenous cysteine for their glutathione synthesis. After application of cysteine to neuron-rich primary cultures, the glutathione content was doubled after a 4-hr incubation. The dipeptide cysteinylglycine (CysGly) was able to substitute for cysteine as exogenous glutathione precursor. In kidneys, the ectopeptidase aminopeptidase N (ApN) has been reported to hydrolyze CysGly. Expression of mRNA of ApN in rat brain and cultured rat neurons was demonstrated by reverse transcriptase polymerase chain reaction and sequencing of the cDNA fragment obtained. In addition, the presence of ApN protein in cultured neurons was demonstrated by its immunocytochemical localization. In the presence of an activity-inhibiting antiserum against ApN the utilization of CysGly as neuronal glutathione precursor was completely prevented, whereas that of cysteine plus glycine was not affected. The data presented demonstrates that cultured rat neurons express ApN and that this ectopeptidase participates in the utilization of CysGly as precursor for neuronal glutathione.