Vulnerability to glutamate toxicity of dopaminergic neurons is dependent on endogenous dopamine and MAPK activation

Dopaminergic neurons are more vulnerable than other types of neurons in cases of Parkinson disease and ischemic brain disease. An increasing amount of evidence suggests that endogenous dopamine plays a role in the vulnerability of dopaminergic neurons. Although glutamate toxicity contributes to the pathogenesis of these disorders, the sensitivity of dopaminergic neurons to glutamate toxicity has not been clarified. In this study, we demonstrated that dopaminergic neurons were preferentially affected by glutamate toxicity in rat mesencephalic cultures. Glutamate toxicity in dopaminergic neurons was blocked by inhibiting extracellular signal-regulated kinase (ERK), c- jun N-terminal kinase, and p38 MAPK. Furthermore, depletion of dopamine by α-methyl- dl - p -tyrosine methyl ester (α-MT), an inhibitor of tyrosine hydroxylase (TH), protected dopaminergic neurons from the neurotoxicity. Exposure to glutamate facilitated phosphoryration of TH at Ser31 by ERK, which contributes to the increased TH activity. Inhibition of ERK had no additive effect on the protection offered by α-MT, whereas α-MT and c- jun N-terminal kinase or p38 MAPK inhibitors had additive effects and yielded full protection. These data suggest that endogenous dopamine is responsible for the vulnerability to glutamate toxicity of dopaminergic neurons and one of the mechanisms may be an enhancement of dopamine synthesis mediated by ERK.     

Modulation of sensory input to the spinal cord by presynaptic ionotropic glutamate receptors

Sensory input from peripheral nerves to the dorsal horn of the spinal cord is mediated by a variety of agents released by the central terminals of dorsal root ganglion (DRG) neurons. These include, but are not limited to, amino acids, especially glutamate, peptides and purines. The unraveling of the mechanisms of synaptic transmission by central terminals of DRG neurons has to take into account various ways in which the message from the periphery can be modulated at the level of the first central synapse. These include postsynaptic and presynaptic mechanisms. Homomeric and heteromeric complexes of receptor subunits for the different transmitters released by DRG neurons and interneurons, clustered at the postsynaptic site of central synapses, can be expressed in different combinations and their rate of insertion into the postsynaptic membrane is activity-regulated. Inhibitory mechanisms are an important part of central modulation, especially via presynaptic inhibition, currently believed to involve GABA released by inhibitory intrinsic neurons. Recent work has established the occurrence of another way by which sensory input can be modulated, i.e. the expression of presynaptic ionotropic and metabotropic receptors in central terminals of DRG neurons. Microscopic evidence for the expression, in these terminals, of various subunits of ionotropic glutamate receptors documents the selective expression of glutamate receptors in functionally different DRG afferents. Electrophysiological and pharmacological data suggest that activation of presynaptic ionotropic glutamate receptors in central terminals of DRG neurons may result in inhibition of release of glutamate by the same terminals. Glutamate activating presynaptic receptors may spill over from the same or adjacent synapses, or may be released by processes of astroglial cells surrounding synaptic terminals. The wide expression of presynaptic ionotropic glutamate receptors, especially in superficial laminae of the dorsal horn, where Adelta- and C fibers terminate, provides an additional or alternative mechanism, besides GABA-mediated presynaptic inhibition, for the modulation of glutamate release by these fibers. Since, however, presynaptic ionotropic glutamate receptors are also expressed in terminals of GABAergic intrinsic interneurons, a decrease of GABA release resulting from activation of these receptors in the same laminae, may also play a role in central sensitization and hyperalgesia.     

Depletion of Intracellular Glutathione Increases Susceptibility to Nitric Oxide in Mesencephalic Dopaminergic Neurons

Using primary neuronal cultures, we investigated the effects of GSH depletion on the cytotoxic effects of glutamate and NO in dopaminergic neurons. Intracellular GSH was depleted by 24-h exposure to L-buthionine-[S,R]-sulfoximine (BSO), an irreversible inhibitor of GSH synthase. BSO exposure caused concentration-dependent reduction of the viability of both dopaminergic and nondopaminergic neurons. In contrast, 24-h exposure of cultures to glutamate or NOC18, an NO-releasing agent, significantly reduced the viability of nondopaminergic neurons without affecting that of dopaminergic neurons. Pretreatment with N-acetyl-L-cysteine for 24 h ameliorated the NOC18-induced toxicity in nondopaminergic neurons. In dopaminergic neurons, sublethal concentrations of BSO reduced intracellular GSH content and markedly potentiated glutamate- and NOC18-induced toxicity. These results suggested that glutamate toxicity was enhanced in dopaminergic neurons by suppression of defense mechanisms against NO toxicity under conditions of GSH depletion. Under such conditions, free iron plays an important role because BSO-enhanced NO toxicity was ameliorated by the iron-chelating agent, deferoxamine. These results suggest that GSH plays an important role in the expression of NO-mediated glutamate cytotoxicity in dopaminergic neurons. Free iron may be related to enhanced NO cytotoxicity under GSH depletion.     

The role of NMDA receptors in the slow neuronal degeneration of Parkinson's disease

Summary Parkinson's disease is a disorder, in which neurons of various neuronal systems degenerate. Furthermore, in such degenerating neurons, the cytoskeleton seems to be affected. In this respect, Parkinson's disease resembles Alzheimer's disease. Since it has been shown, that elevated levels of intracellular calcium can disrupt the cytoskeleton and that the stimulation of glutamate (NMDA) receptors can cause high intracellular concentrations of calcium, it has been suggested, that the stimulation of glutamate receptors plays a role in the slow degeneration in Alzheimer's and Parkinson's disease. In case of the degeneration of the dopaminergic nigrostriatal system in Parkinson's disease, neurons that contain calcium binding protein appear to be less vulnerable than the neurons that lack it, suggesting that calcium binding protein might protect these neurons from degeneration by preventing that cytosolic calcium concentrations increase excessively. And, since there is in the nigrostriatal system a glutamatergic afferent pathway (the prefrontonigral projection) and since dopaminergic nigrostriatal neurons contain postsynaptic NMDA receptors, glutamatergic excitation may play a role in the degeneration of the nigrostriatal system in Parkinson's disease. If so, it may be possible to protect the neurodegeneration of these dopaminergic neurons by NMDA receptor antagonists.     

Interactions between prolactin and dopaminergic neurons

The secretion of prolactin from the adenohypophysis is tonically inhibited by dopamine that is released into the hypophysial portal blood from terminals of tuberoinfundibular neurons located in the external layer of the median eminence. These tuberoinfundibular neurons are unique among other dopaminergic neurons in the brain (including the well-characterized nigrostriatal neurons) in that they are not directly regulated by dopaminergic receptor-mediated mechanisms, but instead are selectively responsive to changes in prolactin concentrations in blood and cerebrospinal fluid. In the rat, the activity of the tuberoinfundibular dopaminergic neurons is higher in the female than in the male, exhibits a characteristic cyclical pattern during the first half of pregnancy and is constantly high as a result of stimulation by placental lactogen during the last 9 days of pregnancy, and is reduced in lactating animals and acutely inhibited during suckling.     

Long-term potentiation of excitatory inputs to brain reward areas by nicotine

Nicotine reinforces smoking behavior by activating nicotinic acetylcholine receptors (nAChRs) in the midbrain dopaminergic (DA) reward centers, including the ventral tegmental area (VTA). Although nicotine induces prolonged excitation of the VTA in vivo, the nAChRs on the DA neurons desensitize in seconds. Here, we show that activation of nAChRs on presynaptic terminals in the VTA enhances glutamatergic inputs to DA neurons. Under conditions where the released glutamate can activate NMDA receptors, long-term potentiation (LTP) of the excitatory inputs is induced. Both the short- and the long-term effects of nicotine required activation of presynaptic alpha7 subunit-containing nAChRs. These results can explain the long-term excitation of brain reward areas induced by a brief nicotine exposure. They also show that nicotine alters synaptic function through mechanisms that are linked to learning and memory.     

Dopaminergic Neurons Respond to Iron-Induced Oxidative Stress by Modulating Lipid Acylation and Deacylation Cycles

Metal-imbalance has been reported as a contributor factor for the degeneration of dopaminergic neurons in Parkinson Disease (PD). Specifically, iron (Fe)-overload and copper (Cu) mis-compartmentalization have been reported to be involved in the injury of dopaminergic neurons in this pathology. The aim of this work was to characterize the mechanisms of membrane repair by studying lipid acylation and deacylation reactions and their role in oxidative injury in N27 dopaminergic neurons exposed to Fe-overload and Cu-supplementation. N27 dopaminergic neurons incubated with Fe (1mM) for 24 hs displayed increased levels of reactive oxygen species (ROS), lipid peroxidation and elevated plasma membrane permeability. Cu-supplemented neurons (10, 50 μM) showed no evidence of oxidative stress markers. A different lipid acylation profile was observed in N27 neurons pre-labeled with [³H] arachidonic acid (AA) or [³H] oleic acid (OA). In Fe-exposed neurons, AA uptake was increased in triacylglycerols (TAG) whereas its incorporation into the phospholipid (PL) fraction was diminished. TAG content was 40% higher in Fe-exposed neurons than in controls. This increase was accompanied by the appearance of Nile red positive lipid bodies. Contrariwise, OA incorporation increased in the PL fractions and showed no changes in TAG. Lipid acylation profile in Cu-supplemented neurons showed AA accumulation into phosphatidylserine and no changes in TAG. The inhibition of deacylation/acylation reactions prompted an increase in oxidative stress markers and mitochondrial dysfunction in Fe-overloaded neurons. These findings provide evidence about the participation of lipid acylation mechanisms against Fe-induced oxidative injury and postulate that dopaminergic neurons cleverly preserve AA in TAG in response to oxidative stress.     

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons

Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U-13C]Glucose and [U-13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of DL-threo-beta-benzyloxyaspartate (TBOA) and in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool.     

Dale's principle and glutamate corelease from ventral midbrain dopamine neurons

Summary. While direct application of dopamine modulates postsynaptic activity, electrical stimulation of dopamine neurons typically evokes excitation. Most of this excitation appears to be due to activation of collateral pathways; however, several lines of evidence have suggested that there is a monosynaptic component due to glutamate corelease by dopamine neurons. Recently, more direct evidence obtained in culture has shown that ventral midbrain dopamine neurons release both dopamine and glutamate. Moreover, they appear to do so from separate release sites, calling into question recent modifications of Dale's Principle. The neurochemical phenotype of a given synapse may be determined by subcellular neurotransmitter levels, uptake, or storage. However, the relationship between dopamine and glutamate release from dopamine neuron synapses in the intact brain – and the mechanisms involved – has yet to be resolved. Received August 31, 1999 Accepted September 20, 1999     

Role of activity-dependent mechanisms in the control of dopaminergic neuron survival

Dopaminergic neurons that constitute the nigrostriatal pathway are characterized by singular electrical properties that allow them to discharge in vivo spontaneously in a spectrum of patterns ranging from pacemaker to random and bursting modes. These electrophysiological features allow dopaminergic neurons to optimize the release of dopamine in their terminal fields. However, there is emerging evidence indicating that electrical activity might also participate in the control of dopaminergic neuron survival, not only during development, but also in the adult brain, thus raising the possibility that alterations in ionic currents could contribute actively to the demise of these neurons in Parkinson disease. This review focuses on the mechanisms by which activity-dependent mechanisms might modulate dopaminergic cell survival.     

Effect of intracerebral administration of NMDA and AMPA on dopamine and glutamate release in the ventral pallidum and on motor behavior

The present study investigates the modulation of the ventral tegmental area (VTA)-ventral pallidum (VP) dopaminergic system by glutamate agonists in rats. The glutamate receptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were infused via reversed microdialysis into the VTA, and dopamine (DA), glutamate, and aspartate levels in the VTA and ipsilateral VP were monitored together with motor behavior screened in an open field. NMDA (750 microM) infusion, as well as AMPA (50 microM) infusion, induced an increase of DA and glutamate levels in the VTA, followed by an increase of DA levels in the ipsilateral VP and by enhanced locomotor activity. The increase of DA in the VP was similar after administration of these two glutamate agonists, although motor activity was more pronounced and showed an earlier onset after NMDA infusion. Glutamate levels in the VP were not increased by the stimulation of DA release. It is concluded that DA is released from mesencephalic DA neurons projecting to the VP and that these neurons are controlled by glutamatergic systems, via NMDA and AMPA receptors. Thus, DA in the VP has to be considered as a substantial modulator. Dysregulation of the mesopallidal DA neurons, as well as their glutamatergic control, may play an additional or distinct role in disorders like schizophrenia and drug addiction.     

Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo

Several people with Parkinson's disease have been treated with intrastriatal grafts of fetal dopaminergic neurons. Following autopsy, 10-22 years after surgery, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. Numerous studies have attempted to explain these findings in cell and animal models. In cell culture, α-synuclein has been found to transfer from one cell to another, via mechanisms that include exosomal transport and endocytosis, and in certain cases seed aggregation in the recipient cell. In animal models, transfer of α-synuclein from host brain cells to grafted neurons has been shown, but the reported frequency of the event has been relatively low and little is known about the underlying mechanisms as well as the fate of the transferred α-synuclein. We now demonstrate frequent transfer of α-synuclein from a rat brain engineered to overexpress human α-synuclein to grafted dopaminergic neurons. Further, we show that this model can be used to explore mechanisms underlying cell-to-cell transfer of α-synuclein. Thus, we present evidence both for the involvement of endocytosis in α-synuclein uptake in vivo, and for seeding of aggregation of endogenous α-synuclein in the recipient neuron by the transferred α-synuclein. Finally, we show that, at least in a subset of the studied cells, the transmitted α-synuclein is sensitive to proteinase K. Our new model system could be used to test compounds that inhibit cell-to-cell transfer of α-synuclein and therefore might retard progression of Parkinson neuropathology.     

P2 receptors are involved in the mediation of motivation-related behavior

The importance of purinergic signaling in the intact mesolimbic–mesocortical circuit of the brain of freely moving rats is reviewed. In the rat, an endogenous ADP/ATPergic tone reinforces the release of dopamine from the axon terminals in the nucleus accumbens as well as from the somatodendritic region of these neurons in the ventral tegmental area, as well as the release of glutamate, probably via P2Y₁ receptor stimulation. Similar mechanisms may regulate the release of glutamate in both areas of the brain. Dopamine and glutamate determine in concert the activity of the accumbal GABAergic, medium-size spiny neurons thought to act as an interface between the limbic cortex and the extrapyramidal motor system. These neurons project to the pallidal and mesencephalic areas, thereby mediating the behavioral reaction of the animal in response to a motivation-related stimulus. There is evidence that extracellular ADP/ATP promotes goal-directed behavior, e.g., intention and feeding, via dopamine, probably via P2Y₁ receptor stimulation. Accumbal P2 receptor-mediated glutamatergic mechanisms seem to counteract the dopaminergic effects on behavior. Furthermore, adaptive changes of motivation-related behavior, e.g., by chronic succession of starvation and feeding or by repeated amphetamine administration, are accompanied by changes in the expression of the P2Y₁ receptor, thought to modulate the sensitivity of the animal to respond to certain stimuli.     

DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T alpha-synuclein toxicity

DJ-1 is the third gene that has been linked to Parkinson disease. Mutations in the DJ-1 gene cause early onset PD with autosomal recessive inheritance. To clarify the mechanism of DJ-1 protection, we have overexpressed the gene in cultured dopaminergic cells that were then subjected to chemical stress. In the rat dopaminergic cell line, N27, and in primary dopamine neurons, overexpression of wild type DJ-1 protected cells from death induced by hydrogen peroxide and 6-hydroxydopamine. Overexpressing the L166P mutant DJ-1 had no protective effect. By contrast, knocking down endogenous DJ-1 with antisense DJ-1 rendered cells more susceptible to oxidative damage. We have found that DJ-1 improves survival by increasing cellular glutathione levels through an increase in the rate-limiting enzyme glutamate cysteine ligase. Blocking glutathione synthesis eliminated the beneficial effect of DJ-1. Protection could be restored by adding exogenous glutathione. Wild type DJ-1 reduced cellular reactive oxygen species and reduced the levels of protein oxidation caused by oxidative stress. By a separate mechanism, overexpressing wild type DJ-1 inhibited the protein aggregation and cytotoxicity usually caused by A53T human alpha-synuclein. Under these circumstances, DJ-1 increased the level of heat shock protein 70 but did not change the glutathione level. Our data indicate that DJ-1 protects dopaminergic neurons from oxidative stress through up-regulation of glutathione synthesis and from the toxic consequences of mutant humanalpha-synuclein through increased expression of heat shock protein 70. We conclude that DJ-1 has multiple specific mechanisms for protecting dopamine neurons from cell death.     

Inflammation and infection in Parkinson's disease

The hallmark of Parkinson's disease (PD) is a specific degeneration of dopaminergic neurons in the substantia nigra (SN). The cause of nigral dopaminergic neuronal cell death in PD and its underlying mechanisms remain elusive, however, involvement of inflammatory events has been postulated because inflammatory features have been described in the brain of PD patients. Some evidence also suggest that a possible deleterious effects of neuroinflammatory processes by infection in experimental models of neurodegenerative disease. In this review, we summarize and discuss the latest findings regarding inflammation in PD. Especially, we focused on the relationship between infection and PD.     

Topical glucose and accumulation of excitotoxic and other amino acids in ischemic cerebral cortex.

Pre-ischemic hyperglycemia aggravates brain damage due to transient global ischemia as demonstrated by exacerbation of brain lesions. Lactacidosis and elevated glutamate levels have been implicated as mechanisms of the increased damage. Our objective was to determine the effects of different levels of glucose (0, 66.5, 450 mg/dL) in cortical superfusates on the ischemia/reperfusion-evoked release of amino acids from the rat cerebral cortex. Physiologic levels of glucose significantly reduced the amount of aspartate, glutamate and gamma-aminobutyric acid and the supra-physiologic levels of glucose reduced the amount of aspartate and phosphoethanolamine released from the cortex during ischemia/reperfusion in comparison with no glucose. The decrease in glutamate release may be due to increased availability of glucose for glycolysis with the subsequent formation of ATP and lactate, which has been shown to act as an energy source for neurons. The decreased levels may also reflect the continued energy-dependent uptake of glutamate by glial cells.     

Mitochondria and dopamine: new insights into recessive parkinsonism

Recessively inherited mutations in parkin, DJ-1, and PINK1 have recently been linked to familial forms of parkinsonism. These syndromes are often clinically indistinguishable from Parkinson's disease, as similar neuronal groups, notably dopaminergic neurons, are selectively affected. Studies of the functions of these gene products may provide insights into the pathogenic mechanisms underlying the selective degeneration of dopaminergic neurons. Emerging evidence that one or several of these genes play important roles in mitochondrial function and the dopaminergic system suggests that these events may be early steps of the pathophysiological changes of the disease. This review will summarize recent advances in our understanding of these gene products, with emphasis on the surprising convergence of their functions.     

Metabolic signaling between neurons and glial cells: a short review.

There is convincing evidence that astrocytes transform blood-born glucose to lactate, alpha-Keto-glutarate and alanine and supply the neurons. There is a tight regulation of this metabolic coupling by means of chemical signals released by functioning neurons. Previous, pioneer, studies have explored several signals-candidates the major being K(+), Ca(++) and several neuromodulators. However, recent results of numerous studies identify glutamate as the major signal that traffics between excited neurons and astrocytes. The excited neurons also produce and release NH(4)(+) in the extracellular space. Both glutamate and ammonium are taken up preferentially by astrocytes and form glutamine. Ammonia fixation by glutamine synthase controls the amount of lactate, glutamine and alanine produced and released by Muller cells in the extracellular space and then taken up by neurons. Thus, there is a tight coupling between function and metabolism in the central neurons system.     

Kainate receptors mediate regulated exocytosis of secretory phospholipase A(2) in SH-SY5Y neuroblastoma cells

Secretory phospholipase A(2) (sPLA(2)) isoforms are widely expressed in the brain and spinal cord. Group IIA sPLA(2) (sPLA(2)-IIA) has been shown to stimulate exocytosis and release of neurotransmitters in neuroendocrine PC12 cells and neurons, suggesting a role of the enzyme in neuronal signaling and synaptic transmission. However, the mechanisms by which sPLA(2) is itself released, and a possible relation between glutamate receptors and sPLA(2) exocytosis, are unknown. This study was carried out to elucidate the effects of glutamate receptor agonists on exocytosis of sPLA(2)-IIA in transfected SH-SY5Y neuroblastoma cells. sPLA(2)-IIA enzyme was packaged in fusion-competent vesicles and released constitutively or upon stimulation, suggesting regulated secretion. The signal peptide of sPLA(2)-IIA is required for its vesicular localization and exocytosis. External application of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) induced vesicular exocytosis and release of sPLA(2)-IIA. UBP 302, a GluR5-specific KA receptor antagonist, abolished the effect of KA, confirming the role of KA receptors in mediating sPLA(2)-IIA secretion. Moreover, KA-induced sPLA(2)-IIA secretion is dependent on Ca(2+) and protein kinase C. Together, these findings provide evidence of a link between glutamate receptors and regulated sPLA(2) secretion in neurons that may play an important role in synaptic plasticity, pain transmission and neurodegenerative diseases.