Antagonizing Nogo-receptor 1 promotes the number of cultured dopaminergic neurons and elongates their neurites

The myelin-associated protein Nogo-A and its receptor Nogo-receptor 1 (NgR1) are known as potent growth inhibitors of the adult central nervous system (CNS). Nogo-A is mostly expressed on the surface of oligodendrocytes, but is also found in neurons of the adult and developing CNS. This observation suggests that Nogo-A serves additional functions in the brain. Hence, in the present study, we investigated the effects of antagonizing NgR1 on cultured organotypic and dissociated dopaminergic neurons. For that purpose ventral mesencephalic cultures from E14 rat embryos were grown in absence or presence of the NgR1 antagonist NEP1-40 for 1 week. Treatment with NEP1-40 significantly increased cell densities of tyrosine hydroxylase-immunoreactive neurons. Moreover, organotypic ventral mesencephalic cultures displayed a significantly bigger volume after NEP1-40 treatment. Morphological analysis of tyrosine hydroxylase-positive neurons disclosed longer neurites and higher numbers of primary neurites in dissociated cultures incubated with NEP1-40, whereas soma size was not changed. In conclusion, our findings demonstrate that interfering with Nogo-A signaling by antagonizing NgR1 modulates dopaminergic neuron properties during development. These observations highlight novel aspects of the role of Nogo-A in the CNS and might have an impact in the context of Parkinson's disease.

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides that are mainly expressed in the gastrointestinal tract. Notably, TFF1 has been suggested to operate as a neuropeptide, however, its specific cellular expression, regulation and function remain largely unknown. We have previously shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10 days in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which could indicate that GDNF and Forskolin targeted different subpopulations of TH/TFF1 neurons. Short-term treatment with Forskolin resulted in an increased number of TFF1-ir cells, and this effect was significantly reduced by the MEK1 inhibitor PD98059 or the protein kinase A (PKA) inhibitor H89, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells.

Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-κB is associated with oxidative stress-induced apoptosis in several nonneuronal in vitro models. To investigate whether it may play a role in PD, we looked for the translocation of NF-κB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-κB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-κB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF-κB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C2-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.

A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons

The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene, Ptx3, a member of the Ptx-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxy-dopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.

Single-channel properties of the nonselective cation conductance induced by neurotensin in dopaminergic neurons

Slow nonselective cation conductances play a central role in determining the excitability of many neurons, but heretofore this channel type has not been analyzed at the single-channel level. Neurotensin (NT) excites cultured dopaminergic neurons from the ventral tegmental area primarily by increasing such a cation conductance. Using the outside–out configuration of the patch clamp, we elicited single-channel activity of this NT-induced cation channel. Channel activity was blocked by the nonpeptide NT antagonist SR48692, indicating that the response was mediated by NT receptors. The channel opened in both solitary form and in bursts. The reversal potential was −4.2 ± 1.7 mV, and the elementary conductance was 31 pS at −67 mV with [Na+]o = 140 mM, [Cs+]o = 5 mM, [Na+]i = 88 mM, and [Cs+]i = 74 mM. Thus, the channel was permeable to both Na+ and Cs+. From these characteristics, it is likely that this channel is responsible for the whole-cell current we studied previously. In guanosine 5′-[γ-thio]triphosphate-loaded cells, NT irreversibly activated about half of the channel activity, suggesting that at least part of the response was mediated by a G protein. Similar channel activity could be induced occasionally in the cell-attached configuration by applying NT outside the patch region.

Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic region of the mouse

Although it has been known for some time that estrogen exerts a profound influence on brain development a definitive demonstration of the role of the classical estrogen receptor (ERα) in sexual differentiation has remained elusive. In the present study we used a sexually dimorphic population of dopaminergic neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) to test the dependence of sexual differentiation on a functional ERα by comparing the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the AVPV of wild-type (WT) mice with that of mice in which the ERα had been disrupted by homologous recombination (ERKOα). Only a few ERα-immunoreactive neurons were detected in the AVPV of ERKOα mice, and the number of TH-immunoreactive neurons was three times that of WT mice, suggesting that disruption of the ERα gene feminized the number of TH-immunoreactive neurons. In contrast, the AVPV contains the same number of TH-immunoreactive neurons in testicular feminized male mice as in WT males, indicating that sexual differentiation of this population of neurons is not dependent on an intact androgen receptor. The number of TH-immunoreactive neurons in the AVPV of female ERKOα mice remained higher than that of WT males, but TH staining appeared to be lower than that of WT females. Thus, the sexual differentiation of dopamine neurons in the AVPV appears to be receptor specific and dependent on the perinatal steroid environment.

Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons

To visualize and isolate live dopamine (DA)-producing neurons in the embryonic ventral mesencephalon, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter. In the transgenic mice, GFP expression was observed in the developing DA neurons containing tyrosine hydroxylase. The outgrowth and cue-dependent guidance of GFP-labeled axons was monitored in vitro with brain culture systems. To isolate DA neurons expressing GFP from brain tissue, cells with GFP fluorescence were sorted by fluorescence-activated cell sorting. More than 60% of the sorted GFP+ cells were positive for tyrosine hydroxylase, confirming that the population had been successfully enriched with DA neurons. The sorted GFP+ cells were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a recovery from Parkinsonian behavioral defects. This strategy for isolating an enriched population of DA neurons should be useful for cellular and molecular studies of these neurons and for clinical applications in the treatment of Parkinson's disease.

Selective vulnerability of late-generated dopaminergic neurons of the substantia nigra in weaver mutant mice.

In homozygous weaver (wv/wv) mutant mice, nearly 50% of the dopaminergic substantia nigra neurons degenerate by postnatal day 20. We have now determined that the total number of dopaminergic neurons in the ventral midbrains of a litter of obligatory homozygous weaver pups and a litter of normal wild-type control pups indicates that no significant differences are present between groups at birth. To test the hypothesis that the subsequent degeneration of these neurons is linked to their time of origin, [3H]thymidine autoradiography was combined with tyrosine hydroxylase immunocytochemistry to construct neurogenetic timetables on postnatal day 20 in wild-type mice and weaver homozygotes. Both groups have the same span of neurogenesis but have statistically different proportions of neurons generated on specific days. In wild-type mice, more than half of the dopaminergic neurons originate on or after embryonic day 12. In contrast, over two-thirds of the surviving dopaminergic neurons in homozygous weaver mice originate on or before embryonic day 11. Our data suggest that the weaver gene does not interfere with the generation of dopaminergic neurons, but it preferentially kills late-generated dopaminergic neurons between birth and postnatal day 20.

Glial cell line-derived neurotrophic factor but not transforming growth factor beta 3 prevents delayed degeneration of nigral dopaminergic neurons following striatal 6-hydroxydopamine lesion.

Glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor beta 3 (TGF-beta 3) are members of the TGF-beta superfamily with high neurotrophic activity on cultured nigral dopamine neurons. We investigated the effects of intracerebral administration of GDNF and TGF-beta 3 on the delayed cell death of the dopamine neurons in the rat substantia nigra following 6-hydroxydopamine lesions of dopaminergic terminals in the striatum. Fluorescent retrograde tracer injections and tyrosine hydroxylase immunocytochemistry demonstrated nigral degeneration with an onset 1 week after lesion, leading to extensive death of nigral neurons 4 weeks postlesion. Administration of recombinant human GDNF for 4 weeks over the substantia nigra at a cumulative dose of 140 micrograms, starting on the day of lesion, completely prevented nigral cell death and atrophy, while a single injection of 10 micrograms 1 week postlesion had a partially protective effect. Continuous administration of TGF-beta 3, starting on the day of lesion surgery, did not affect nigral cell death or atrophy. These findings support the notion that GDNF, but not TGF-beta 3, is a potent neurotrophic factor for nigral dopamine neurons in vivo.