Effects of glutamate antagonists on the activity of aromatic L-amino acid decarboxylase

Summary This study examines the hypothesis that glutamate tonically suppresses the activity of the enzyme aromatic L-amino acid decarboxylase (AADC), and hence the biosynthesis of dopamine, to explain how antagonists of glutamate receptors might potentiale the motor actions of L-DOPA in animal models of Parkinson's disease. A variety of glutamate antagonists were therefore administered acutely to normal rats, which were sacrificed 30–60 min later and AADC activity assayed in the substantia nigra pars reticulate (SNr) and corpus striatum (CS). The NMDA receptor-ion channel antagonists MK 801, budipine, amantadine, memantine and dextromethorphan all caused a pronounced in creased in AADC activity, more especially in the SNr than CS. The NMDA glycine site antagonist (R)-HA 966 produced a modest increase in AADC activity in the CS but not SNr, whilst the NMDA polyamine site antagonist eliprodil, the NMDA competitive antagonist CGP 40116 and the AMPA antagonist NBQX were without effect. The results suggest that an increase in dopamine synthesis might contribute to the L-DOPA-facilitating actions of some glutamate antagonists.     

Alpha-synuclein inhibits aromatic amino acid decarboxylase activity in dopaminergic cells

Alpha-synuclein is a presynaptic protein strongly implicated in Parkinson's disease (PD). Because dopamine neurons are invariably compromised during pathogenesis in PD, we have been exploring the functions of alpha-synuclein with particular relevance to dopaminergic neuronal cells. We previously discovered reduced tyrosine hydroxylase (TH) activity and minimal dopamine synthesis in stably-transfected MN9D cells overexpressing either wild-type or A53T mutant (alanine to threonine at amino acid 53) alpha-synuclein. TH, the rate-limiting enzyme in dopamine synthesis, converts tyrosine to l-dihydroxyphenylalanine (L-DOPA), which is then converted to dopamine by the enzyme, aromatic amino acid decarboxylase (AADC). We confirmed an interaction between alpha-synuclein and AADC in striatum. We then sought to determine whether wild-type or A53T mutant alpha-synuclein might have affected AADC activity in dopaminergic cells. Using HPLC with electrochemical detection, we measured dopamine and related catechols after L-DOPA treatments to bypass the TH step. We discovered that while alpha-synuclein did not reduce AADC protein levels, it significantly reduced AADC activity and phosphorylation in our cells. These novel findings further support a role for alpha-synuclein in dopamine homeostasis and may explain, at least in part, the selective vulnerability of dopamine neurons that occurs in PD.     

Long-Term L-DOPA Treatment Causes Indiscriminate Increase in Dopamine Levels at the Cost of Serotonin Synthesis in Discrete Brain Regions of Rats

(1) The treatment of choice for Parkinson’s disease (PD) is 3,4-dihydroxyphenylalanine (L-DOPA) with peripheral decarboxylase inhibitor, but long-term therapy leads to motor and psychiatric complications. In the present study we investigated 5-hydroxytryptamine (5-HT) and dopamine concentrations in serotonergic and dopaminergic nuclei following chronic administration of L-DOPA to find whether the neurotransmitter synthesis in these brain areas are compensated. (2) Rats were administered L-DOPA (250 mg/kg) and carbidopa (25 mg/kg) daily for 59 and 60 days, and killed on the 60th day, respectively at 24 h and 30 min after the last dose. L-DOPA, norepinephrine, 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), dopamine, homovanillic acid (HVA), and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in striatum, nucleus raphe dorsalis (NRD), nucleus accumbens (NAc), substantia nigra, cerebellum, and cortex employing HPLC-electrochemical procedure. (3) Prolonged treatment of L-DOPA caused depression in the animals as revealed in a forced swim test. Serotonin content was significantly decreased in all brain regions studied 30 min after long-term L-DOPA, except in NAc. The cortex and striatum showed lowered levels of this indoleamine 24 h after 59 doses of L-DOPA. Dopamine, HVA, and DOPAC concentrations were significantly higher in all the regions studied after 30 min, and in the cerebellum after 24 h of L-DOPA. The levels of DOPAC were elevated in all the brain areas studied 24 h after prolonged L-DOPA treatment. (4) The present results suggest that long-term L-DOPA treatment results in significant loss of 5-HT in serotonergic and dopaminergic regions of the brain. Furthermore, while L-DOPA metabolism per se was uninfluenced, dopamine synthesis was severely impaired in all the regions. The imbalance of serotonin and dopamine formation may be the cause of overt cognitive, motor, and psychological functional aberrations seen in parkinsonian patients following prolonged L-DOPA treatment.     

L-DOPA does not enhance hydroxyl radical formation in the nigrostriatal dopamine system of rats with a unilateral 6-hydroxydopamine lesion

The debate about the toxicity of L-DOPA to dopaminergic neurons has not been resolved. Even though enzymatic and nonenzymatic metabolism of L-DOPA can produce hydrogen peroxide and oxygen free radicals, there has been controversy as to whether L-DOPA generates an oxidant stress in vivo. This study determined whether acute or repeated administration of L-DOPA caused in vivo production of hydroxyl radicals in striatum and other brain regions in rats with a unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal projections. Salicylate trapping combined with in vivo microdialysis provided measurements of extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA) in striatum following L-DOPA administration systemically (100 mg/kg, i.p.) or by intrastriatal perfusion (1 mM, via the microdialysis probe). Tissue concentrations of 2,3-DHBA and salicylate were also measured in striatum, ventral midbrain, and cerebellum following repeated administration of L-DOPA (50 mg/kg, i.p., once daily for 16 days). In each instance, treatment with L-DOPA did not increase 2,3-DHBA concentrations, regardless of the nigrostriatal dopamine system's integrity. When added to the microdialysis perfusion medium, L-DOPA resulted in a significant decrease in the striatal extracellular concentration of 2,3-DHBA. These results suggest that administration of L-DOPA, even at high doses, does not induce hydroxyl radical formation in vivo and under some conditions may actually diminish hydroxyl radical activity. Furthermore, prior damage to the nigrostriatal dopamine system does not appear to predispose surviving dopaminergic neurons to increased hydroxyl radical formation following L-DOPA administration. Unlike L-DOPA, systemic administration of methamphetamine (10 mg/kg, s.c.) produced a significant increase in the concentration of 2,3-DHBA in striatal dialysate, suggesting that increased formation of hydroxyl radicals may contribute to methamphetamine neurotoxicity.     

Cysteine and mercapturate conjugates of oxidized dopamine are in human striatum but only the cysteine conjugate impedes dopamine trafficking in vitro and in vivo

Recent results have suggested that some products of mercapturic acid pathway (MAP) metabolism of oxidized dopamine (DA) may contribute to mesostriatal dopaminergic neurodegeneration, and that at least one product, 5-S-cysteinyldopamine (Cys-DA), is elevated in patients with advanced Parkinson's disease (PD) who have been treated with L-DOPA. Here we investigated MAP enzymes and products in the midbrain and striatum of control individuals and patients with dementia with Lewy bodies (DLB) who had less severe dopaminergic degeneration than PD patients and who were not treated with L-DOPA. We also determined the biological activity of MAP metabolites of oxidized DA using primary rat mesencephalic cultures, rat cerebral synaptosomes, and rat striatum in vivo microdialysis. Our results showed that the human mesostriatal dopaminergic pathway generates Cys-DA but has limited enzymatic capacity for mercapturate formation, that striatal levels of MAP products of oxidized DA are not elevated in DLB patients compared with controls, and that Cys-DA interferes with trafficking of DA in vitro and in vivo. These results indicate that while Cys-DA is not increased in striatum of patients with mild dopaminergic neurodegeneration, it may interfere with uptake of DA in patients with advanced PD.     

The effect of intrastriatal injection of liposome-entrapped tyrosinase on the dopamine levels in the rat brain.

Parkinson's disease is a neurodegenerative disorder which is mainly characterized by degeneration of the dopaminergic cells in the nigro-striatal system. Due to a lowered L-tyrosine 3-monooxygenase activity, L-tyrosine is not sufficiently transformed to L-DOPA. To date the most common therapy is the administration of the dopamine precursor L-DOPA, with severe collateral effects. Therefore, the substitution of the lacking tyrosine hydroxylase with tyrosinase might be a novel therapeutical approach that would generate specifically L-DOPA from L-tyrosine. We present here evidence that stereotaxic injection of liposome-entrapped tyrosinase is able to significatively increase the levels of dopamine in the rat brain. The catecholamines L-DOPA, dopamine, L-epinephrine, L-norepinephrine were extracted by acid treatment from the brains and detected by HPLC.     

Adeno-associated virus-mediated gene transfer of human aromatic L-amino acid decarboxylase protects mixed striatal primary cultures from L-DOPA toxicity

Although L-DOPA is the drug of choice for Parkinson's disease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV-hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson's disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high concentrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofilaments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV-hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cultured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity.     

Dopamine Sulfoconjugation in the Rat Brain: Regulation by Monoamine Oxidase

An increase of free 3,4-dihydroxyphenylethylamine (DA, dopamine) in the rat brain such as is found following 3,4-dihydroxyphenylalanine (L-DOPA) administration or an intraventricular injection of free dopamine did not result in DA sulfate formation, despite the presence of phenolsulfotransferase activity in various regions of the brain and the high affinity of DA for this enzyme. However, when rats were pretreated with pargyline, a monoamine oxidase inhibitor, the same treatment with L-DOPA or free DA led to active synthesis of DA sulfate. The increase in DA sulfate was significantly correlated with the degree of monoamine oxidase inhibition and directly proportional to free DA concentrations in the hypothalamus (r = 0.86), striatum (r = 0.54), and brainstem (r = 0.89). The highest ratio of DA sulfate to free DA was found in the hypothalamus, suggesting that sulfoconjugation is most active in this region. Prior treatment of rats with 6-hydroxydopamine did not decrease DA sulfate concentrations, indicating that sulfoconjugation occurs most likely in extraneuronal tissues not destroyed by the neurotoxin. The results are compatible with the notion that phenolsulfotransferase may be highly compartmentalized and that inhibition of monoamine oxidase allows the newly generated free DA to become accessible to the sulfoconjugating enzyme, resulting in increase in DA sulfation.     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

Reserpine pretreatment prevents increases in extracellular striatal dopamine following L-DOPA administration in rats with nigrostriatal denervation

The influence of L-DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L-DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine-pretreated rats. In rats with 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L-DOPA (50 mg/kg i.p.). In 6-hydroxydopamine-lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L-DOPA (50 mg/kg i.p.)-induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p<0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L-DOPA-derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.     

Amino Acids and Biogenic Amines in Cerebrospinal Fluid of Patients with Parkinson's Disease

To study changes in amino acid metabolism and biogenic amines in Parkinson's disease, we set up a prospective study and measured biogenic amines, their main metabolites, and 22 different amino acids, in cerebrospinal fluid of Parkinson's disease patients (n = 24) and age-matched controls (n = 30). A trend toward higher dopamine levels in Parkinson's disease patients was interpreted as an effect of treatment with levodopa and/or selegiline. Significantly lower concentrations of the dopamine metabolite 3,4-dihydroxyphenylacetic acid in the Parkinson's disease group might reflect dopaminergic cell loss. Our results revealed decreased serotonin catabolism that was interpreted as an effect of treatment with selegiline. Whereas all amino acid levels were unchanged, taurine was significantly lower in Parkinson's disease patients. Studies showed that taurine exerts a trophic action on the central nervous system. In this view, decreased taurine in a neurodegenerative disorder as Parkinson's disease deserves attention.     

Protection of intracellular dopamine cytotoxicity by dopamine disposition and metabolism factors

Dopamine has been hypothesized as a contributing factor for the selective degeneration of dopaminergic neurons in Parkinson's disease. However, the cytotoxic mechanisms of dopamine and its metabolites remain poorly understood. Using a stable aromatic amino acid decarboxylase (AADC) expressing a fibroblast cell line, we previously demonstrated a novel, non-oxidative cytotoxicity of intracellular dopamine. In this study, we further investigate the roles of dopamine metabolism and disposition proteins against intracellular dopamine cytotoxicity by co-expressing these factors in AADC-expressing cells. Our results indicate that overexpression of the vesicular monoamine transporter and monoamine oxidase A-induced protection against intracellular dopamine toxicity, and conversely that pharmacological inhibition of these pathways potentiated L-DOPA toxicity in catecholaminergic PC12 cells. Macrophage migration inhibitory factor and glutathione S-transferase (GST), factors that have recently been shown to be involved in dopamine metabolism, also exhibited a strong protective role against intracellular dopamine cytotoxicity. Our results support a potential role for non-oxidative cytoplasmic dopamine toxicity, and imply that disruption in dopamine disposition and/or metabolism could underlie the progressive degeneration of dopaminergic neurons in Parkinson's disease.     

EFFECTS OF HISTIDINE ON THE TRANSPORT OF L-DOPA BY SLICES OF BRAIN CORTEX AND STRIATUM OF THE RAT

Abstract— L-Histidine inhibits the uptake of L-DOPA by brain cortex or striatum slices in short-term experiments, but increases it in long-term experiments. The potentiation of the steady-state accumulation of L-DOPA by histidine is a result of the cis- and trans -effects of histidine on the transport of L-DOPA at both sides of the cell membrane. The results demonstrate the importance of hetero-exchange phenomena in determining the cellular level attained for a given amino acid at the steady-state.     

Gene therapy for Parkinson's disease: determining the genes necessary for optimal dopamine replacement in rat models.

This article reviews the mechanism of dopamine delivery in the CNS in order to determine the optimal set of genes for effective gene therapy in Parkinson's disease (PD). Systematic neurobiological investigation of the biochemical steps has revealed that tyrosine hydroxylase (TH), which has been used in earlier studies, functions only when the essential cofactor, tetrahydrobiopterin (BH1) is present. Transduction of the gene for GTP cyclohydrolase I, the first and rate-limiting step in BH1 synthesis, along with the TH gene, generated cells that are capable of producing L-DOPA spontaneously both in vitro and in vivo. When the aromatic L-amino acid decarboxylase (AADC) gene was added as a third gene, in an attempt to increase the conversion of L-DOPA to dopamine, feedback inhibition by the end product, dopamine, on TH activity resulted. To circumvent this problem, we employed a complementary strategy. Gene transfer of the vesicular monoamine transporter was combined with AADC and produced genetically modified cells that can convert L-DOPA to dopamine and store it for gradual release. This approach provided a means to regulate final dopamine delivery by controlling precursor doses and to achieve more sustained delivery of dopamine. Our investigation into determining the genes necessary for optimal dopamine delivery has been facilitated by in vivo biochemical assays using microdialysis. This technique has provided us with a clear and quantitative tool to compare the effects of various genes involved in dopamine synthesis and processing.     

Bromocriptine Markedly Suppresses Levodopa-Induced Abnormal Increase of Dopamine Turnover in the Parkinsonian Striatum

Bromocriptine, a dopamine agonist, is commonly used in combination with levodopa for the treatment of Parkinson's disease (PD). To investigate the theoretical basis of such combination therapy, we examined the effects of bromocriptine administered alone or in combination with levodopa on dopamine turnover in the striatum of hemi-parkinsonism rats. The parkinsonian striatum showed a 3.4-fold increase of dopamine turnover relative to the control striatum, as often observed in the brain of PD patients. A 7-day course of levodopa therapy markedly increased dopamine turnover in the parkinsonian striatum (53-fold of control level) than in the control striatum (5-fold of the control level). However, bromocriptine specifically and markedly suppressed the levodopa-induced abnormal activation of dopamine turnover in the parkinsonian striatum. Our findings explain the pharmacological basis for the introduction of bromocriptine during long-term levodopa therapy.     

Neuroprotective and neurotoxic roles of levodopa (L-DOPA) in neurodegenerative disorders relating to Parkinson's disease

Summary.  Despite its being the most efficacious drug for symptom reversal in Parkinson's disease (PD), there is concern that chronic levodopa (L-DOPA) treatment may be detrimental. In this paper we review the potential for L-DOPA to 1) autoxidize from a catechol to a quinone, and 2) generate other reactive oxygen species (ROS). Overt toxicity and neuroprotective effects of L-DOPA, both in vivo and in vitro, are described in the context of whether L-DOPA may accelerate or delay progression of human Parkinson's disease. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Striatal Astrocytes Act as a Reservoir for L-DOPA

L-DOPA is therapeutically efficacious in patients with Parkinson’s disease (PD), although dopamine (DA) neurons are severely degenerated. Since cortical astrocytes express neutral amino acid transporter (LAT) and DA transporter (DAT), the uptake and metabolism of L-DOPA and DA in striatal astrocytes may influence their availability in the dopaminergic system of PD. To assess possible L-DOPA- and DA-uptake and metabolic properties of striatal astrocytes, we examined the expression of L-DOPA, DA and DAT in striatal astrocytes of hemi-parkinsonian model rats after repeated L-DOPA administration, and measured the contents of L-DOPA, DA and their metabolite in primary cultured striatal astrocytes after L-DOPA/DA treatment. Repeated injections of L-DOPA induced apparent L-DOPA- and DA-immunoreactivities and marked expression of DAT in reactive astrocytes on the lesioned side of the striatum in hemi-parkinsonian rats. Exposure to DA for 4h significantly increased the levels of DA and its metabolite DOPAC in cultured striatal astrocytes. L-DOPA was also markedly increased in cultured striatal astrocytes after 4-h L-DOPA exposure, but DA was not detected 4 or 8h after L-DOPA treatment, despite the expression of aromatic amino acid decarboxylase in astrocytes. Furthermore, the intracellular level of L-DOPA in cultured striatal astrocytes decreased rapidly after removal of extracellular L-DOPA. The results suggest that DA uptaken into striatal astrocytes is rapidly metabolized and that striatal astrocytes act as a reservoir of L-DOPA that govern the uptake or release of L-DOPA depending on extracellular L-DOPA concentration, but are less capable of converting L-DOPA to DA.     

Dopamine release from serotonergic nerve fibers is reduced in L-DOPA-induced dyskinesia

L-DOPA is the most commonly used treatment for symptomatic control in patients with Parkinson's disease. Unfortunately, most patients develop severe side-effects, such as dyskinesia, upon chronic l-DOPA treatment. The patophysiology of dyskinesia is unclear; however, involvement of serotonergic nerve fibers in converting l-DOPA to dopamine has been suggested. Therefore, potassium-evoked dopamine release was studied after local application of l-DOPA in the striata of normal, dopamine- and dopamine/serotonin-lesioned l-DOPA na?ve, and dopamine-denervated chronically l-DOPA-treated dyskinetic rats using in vivo chronoamperometry. The results revealed that local l-DOPA administration into normal and intact hemisphere of dopamine-lesioned l-DOPA na?ve animals significantly increased the potassium-evoked dopamine release. l-DOPA application also increased the dopamine peak amplitude in the dopamine-depleted l-DOPA na?ve striatum, although these dopamine levels were several-folds lower than in the normal striatum, whereas no increased dopamine release was found in the dopamine/serotonin-denervated striatum. In dyskinetic animals, local l-DOPA application did not affect the dopamine release, resulting in significantly attenuated dopamine levels compared with those measured in l-DOPA na?ve dopamine-denervated striatum. To conclude, l-DOPA is most likely converted to dopamine in serotonergic nerve fibers in the dopamine-depleted striatum, but the dopamine release is several-fold lower than in normal striatum. Furthermore, l-DOPA loading does not increase the dopamine release in dyskinetic animals as found in l-DOPA na?ve animals, despite similar density of serotonergic innervation. Thus, the dopamine overflow produced from the serotonergic nerve fibers appears not to be the major cause of dyskinetic behavior.     

Aging Produces a Specific Pattern of Striatal Dopamine Loss: Implications for the Etiology of Idiopathic Parkinson''s Disease

To examine the possible causal contribution of normal or accelerated aging to the neurodegenerative process of Parkinson's disease, we measured the influence of aging on subregional striatal dopamine and homovanillic acid levels in postmortem brain of 23 neurologically and psychiatrically normal human subjects 14-92 years old. We observed a significant decline in striatal dopamine levels and increase in the homovanillic acid/dopamine molar ratios with increasing age. The dopamine loss, on average, was of the same magnitude in the caudate nucleus and the putamen (-60% in the 84-year-old group as compared with the 22-year-old group), with the caudal component of both nuclei being more affected than the rostral subdivisions. The level of subregional dopamine metabolism, as measured by the homovanillic acid/dopamine ratio, in our young individuals (mean age, 22 years) was found to be inversely correlated to the degree of subregional dopamine loss suffered by the individuals in the older age groups. We conclude the following: (a) Striatal subdivisions with physiologically higher dopamine metabolism are not at a greater risk of suffering dopamine neuronal damage with advancing age, as would seem to be implied by the oxidative stress hypothesis; thus, formation of dopamine-derived oxy radicals in the human striatum appears unlikely to be a primary factor responsible for the age-related striatal dopamine loss. (b) The regional and subregional pattern of striatal dopamine loss in normal aging differs substantially from the pattern typically observed in idiopathic Parkinson's disease; therefore, the cause of idiopathic Parkinson's disease cannot be primarily an age-dependent neurodegenerative process.     

Increased levels of ubiquitin in the 6-OHDA-lesioned striatum of rats

Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar Parkinson's disease. We therefore examined the possible role of 6-hydroxydopamine, a dopaminergic neurotoxin used in Parkinson's disease experimental models, in causing protein degradation and its association with the ubiquitin proteasome system. Using unilaterally 6-hydroxydopamine-denervated rats and mass spectrometry profiling directly on brain tissue sections, we here report for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted hemisphere. No similar changes were found in the intact hemisphere or in the ventral striatum of the dopamine depleted hemisphere. The lesioning of the dopamine innervation to the striatum was confirmed by a strongly reduced dopamine transporter binding in the striatum, indicating an abundant loss of dopamine neurons. These results suggest that denervation of dopamine neurons per se is implicated in the regulation of ubiquitin pathways, at least in a classical animal model of Parkinson's disease. This study adds additional information regarding the involvement of the ubiquitin-proteasome system in Parkinson's disease.