Epigenetic factors and midbrain dopaminergic neurone development

In the mammalian brain dopamine systems play a central role in the control of movement, hormone release, emotional balance and reward. Alteration of dopaminergic neurotransmission is involved in Parkinson's disease and other movement disorders, as well as in some psychotic syndromes. This review summarises recent findings, which shed some light on signals and cellular interactions involved in the specification and maturation of the dopaminergic function during neurogenesis. In particular we will focus on three major issues: (1) the differentiation of dopaminergic neurones triggered by direct contact with the midbrain floor plate cells through the action of sonic hedgehog; (2) the neurotrophic factors acting on dopaminergic neurones; and (3) the role of target striatal cells on the survival and the axonal growth of developing or grafted dopaminergic neurones.     

Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression

In vitro expanded CNS precursors could provide a renewable source of dopamine (DA) neurons for cell therapy in Parkinson's disease. Functional DA neurons have been derived previously from early midbrain precursors. Here we demonstrate the ability of Nurr1, a nuclear orphan receptor essential for midbrain DA neuron development in vivo, to induce dopaminergic differentiation in naïve CNS precursors in vitro. Independent of gestational age or brain region of origin, Nurr1‐induced precursors expressed dopaminergic markers and exhibited depolarization‐evoked DA release in vitro. However, these cells were less mature and secreted lower levels of DA than those derived from mesencephalic precursors. Transplantation of Nurr1‐induced DA neuron precursors resulted in limited survival and in vivo differentiation. No behavioral improvement in apomorphine‐induced rotation scores was observed. These results demonstrate that Nurr1 induces dopaminergic features in naïve CNS precursors in vitro. However, additional factors will be required to achieve in vivo function and to unravel the full potential of neural precursors for cell therapy in Parkinson's disease.     

The beta-chemokines CCL2 and CCL7 are two novel differentiation factors for midbrain dopaminergic precursors and neurons

β-chemokines are secreted factors that regulate diverse functions in the adult brain, such as neuro-immune responses and neurotransmission, but their function in the developing brain is largely unknown. We recently found that the orphan nuclear receptor, Nurr1, up regulates CCL2 and CCL7 in neural stem cells, suggesting a possible function of β-chemokines in midbrain development. Here we report that two β-chemokines, CCL2 and CCL7, and two of their receptors, CCR1 and CCR2, are expressed and developmentally regulated in the ventral midbrain (VM). Moreover, we found that the expression of CCL7 was down regulated in the Nurr1 knockout mice, linking CCL7 to dopamine (DA) neuron development. When the function of CCL2 and CCL7 was examined, we found that they selectively enhanced the differentiation of Nurr1+ precursors into DA neurons, but not their survival or progenitor proliferation in primary precursor cultures. Moreover, both CCL2 and CCL7 promoted neuritogenesis in midbrain DA neuron cultures. Thus, our results show for the first time a function of β-chemokines in the developing brain and identify β-chemokines as novel class of pro-differentiation factors for midbrain DA neurons. These data also suggest that β-chemokines may become useful tools to enhance the differentiation of DA cell preparations for cell replacement therapy and drug discovery in Parkinson's disease (PD).     

Modulatory Effect of Dopamine on High-Affinity Glutamate Uptake in the Rat Striatum

In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium-dependent high-affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10(-8) M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. alpha-Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating effects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6-hydroxydopamine (6-OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6-OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.     

Expression patterns of Nurr1 in rat retina development

Nurr1 is a member of the nuclear receptor superfamily, and is involved in regulating the differentiation, migration and maturation of mesencephalic dopaminergic neurons. The present study was designed to observe Nurr1 protein expression patterns during rat retina development. Immunohistochemical double staining, fluorescence double staining and western blotting were used. The results revealed dramatic and dynamic changes in Nurr1 protein expression during retinal development. Nurr1-positive cells appeared in small quantities at embryonic day 18, and their number then increased markedly during development. The peak occurred at postnatal days 3–7. As maturation continued, the number of positive cells gradually decreased. Comparative observation of Nurr1 and PCNA showed that Nurr1 was confined to differentiated and migrating immature cells, and that it was not present in proliferating cells. Nurr1-positive cells, identified by comparative observation of Nurr1 and syntaxin-1, were amacrine cells. In addition, the Nurr1 and tyrosine hydroxylase coexisted in the same cells, but most cells with Nurr1 expression did not express tyrosine hydroxylase. These results suggest that Nurr1 may play a regulatory role in the differentiation and maturation of both dopaminergic and non-dopaminergic amacrine cells in the rat retina.     

Inhibitory effect of nitric oxide on dopamine transporters: interneuronal communication without receptors

Previously we observed that Nomega-nitro-L-arginine methyl ester (l-NAME) decreased the striatal dopamine (DA) release in microdialysis experiments and this effect was completely diminished in the presence of the DA uptake inhibitor nomifensine, indicating that the effect was mediated via the DA transporter. The aim of the present work was to study the direct effect of nitrergic compounds on DA uptake. We measured the uptake of [3H]DA in striatal slices and found that the nitric oxide (NO) generator sodium nitroprussid (100 microM) decreased the uptake by 66%. In contrast, the NO synthase inhibitor L-NAME (100 microM) increased the DA uptake by 80%, while the inactive D-NAME had no effect on uptake. Our data indicate that NO exerts an inhibitory effect on DA transporters. Since the production of NO by neuronal NO synthase is closely related to the activation of NMDA receptors, the level of NO around synapses reflects the activity of glutamatergic neurotransmission. The strength of excitatory input, therefore, can be nonsynaptically signaled by NO to the surrounding dopaminergic neurons via the inhibitory tone on transporters. The concomitant elevation of DA concentration around the activated synapse represents the response of dopaminergic system, which can adapt to the changing excitatory activity without receiving glutamatergic input and without expressing glutamate receptors. Thus, the effect of NO on transporters represents a new form of interneuronal communication, a nonsynaptic interaction without receptors.     

The role of alpha-synuclein in the development of the dopaminergic neurons in the substantia nigra and ventral tegmental area

Alpha-synuclein is a presynaptic protein of vertebrates that belongs to the family of synucleins. Normal functions of synucleins remain unknown. Alpha-synuclein is one of the causative factors of the familial and idiopathic forms of Parkinson’s disease (PD). The progressive loss of dopaminergic (DA) neurons is characteristic of PD and the most severe damage occurs in the substantia nigra (SN). This leads to an erraticism of the synthesis and synaptic secretion of the neurotransmitters, subsequently resulting in the loss of the connections between brain areas. This work shows that alpha-synuclein is directly involved in the formation of the mature DA neurons of the midbrain at different stages of the ontogenesis and these findings are consistent with data obtained in other studies. Thus, alpha-synuclein may have a varying modulating effect on the growth dynamics and the fate of populations of DA neurons.     

3H-xylamine binds to presynaptic rat striatal membranes

3H-xylamine (3H-XYL), an irreversible catecholamine uptake inhibitor, was incubated with rat striatal synaptosomes, and the membrane fraction was examined by fluorography of a sodium dodecyl sulfate-polyacrylamide gel. A number of peptides were labeled. To determine their location, the striatal dopaminergic presynaptic nerve terminals were destroyed by unilateral electrolytic lesions through the nigrostriatal fibers prior to 3H-XYL exposure. The 3H-XYL bound to membranes from lesioned striata was about 29% of that bound to control membranes, which is consistent with the 83% reduction in dopamine (DA) uptake and the 68% reduction in DA content in the lesioned tissue. The decrease in peptide-bound 3H-XYL paralleled the decrease in DA content, with the exception of a 45% decrease in binding to a 45K peptide. These data show that 3H-XYL binding is predominantly localized in the dopaminergic presynaptic nerve terminals of the striatum.