Erythropoietin and bone morphogenetic protein 7 mediate ascorbate-induced dopaminergic differentiation from embryonic mesencephalic precursors

Mesencephalic precursors derived from early (embryonic day 12; E12) rat embryos were grown in vitro using mitogen basic fibroblast growth factor (bFGF) and these cells efficiently differentiated into dopaminergic (DA) neurons. However, this in vitro DA differentiation was poor in mesencephalic precursors isolated from later embryos (E13-15). Ascorbate (AA) treatment enhanced yields of DA neurons from E12 precursors, and increased the number of DA neurons generated from E13 precursors to levels attained when using E12 precursors. AA markedly up-regulated expression of bone morphogenetic protein 7 (BMP7) and erythropoietin (Epo) in precursors, but did not affect expression of a number of genes known to regulate midbrain DA development. The addition of these recombinant proteins or blockers revealed that both BMP7 and Epo mediate AA-induced DA neuron differentiation.     

Ascorbate-induced differentiation of embryonic cortical precursors into neurons and astrocytes

A specific role for ascorbate (AA) in brain development has been postulated based on a rise of AA levels in fetal brain (Kratzing et al., 1985). To evaluate the role of AA during CNS development, we analyzed the survival, proliferation, and differentiation of AA-treated CNS precursor cells isolated from rat embryonic cortex. Immunocytochemical analyses revealed that AA promoted the in vitro differentiation of CNS precursor cells into neurons and astrocytes in a cell density-dependent manner. Additionally, AA increased the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) of postmitotic neurons in primary neuronal cultures. Differential expression analysis of genes specific to neuronal or glial differentiation revealed an AA-dependent increase in the expression of genes that could potentially compound the effects of AA on cell differentiation. These data suggest that AA may act in the developing brain to stimulate the generation of CNS neurons and glia, thereby assisting in the formation of neural circuits by promoting the acquisition of neuronal synaptic functions.     

Protection from 1-methyl-4-phenylpyridinium (MPP) toxicity and stimulation of regrowth of MPP-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF

Several peptide growth factors can maintain survival or promote recovery of injured central neurons. In the present study, the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the toxicity produced by the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), were investigated in rat mesencephalic dopaminergic neurons in culture. High affinity [3H]DA uptake and morphometric analyses of tyrosine hydroxylase immunostained neurons were used to assess the extent of MPP+ toxicity, dopaminergic neuronal survival and growth of neurites. Consistent with previous reports, EGF and bFGF treatments stimulated neuritic outgrowth in dopaminergic neurons, increased DA uptake and enhanced their long-term survival in vitro. These growth factors also stimulated proliferation of astrocytes. The time course of EGF and bFGF effects on dopaminergic neurons coincided with the increase in glial cell density, suggesting that proliferation of glia mediates their trophic effects. Several findings from our study support this possibility. When MPP+ was applied to cultures at 4 days in vitro, before glial cells had proliferated, the damage to dopaminergic neurons was not affected by EGF or bFGF pretreatments. However, when cultures maintained in the presence of the growth factors for 10 days were exposed to MPP+, after they had become confluent with dividing glial cells, the MPP(+)-induced decreases in DA uptake and cell survival were significantly attenuated. Furthermore, when glial cell proliferation was inhibited by 5-fluoro-2'-deoxyuridine, the protective effects of EGF and bFGF against MPP+ toxicity were abolished. Continuous treatment of MPP(+)-exposed cultures with EGF or bFGF resulted in the stimulation of process regrowth of damaged dopaminergic neurons with concomitant recovery of DA uptake, suggesting that the injured neurons are able to respond to the trophic effects of EGF and bFGF. In summary, our study shows that the trophic effects of EGF and bFGF on mesencephalic dopaminergic neurons include protection from the toxicity produced by MPP+ and promotion of recovery of MPP(+)-damaged neurons. Stimulation of glial cell proliferation is necessary for these effects.     

The transcription factor Nurr1 in human NT2 cells and hNT neurons

Human, neuronally committed hNT or NT2-N cells, originally derived from the Ntera2/D1 (NT2) clone after exposure to retinoic acid (RA), represent a potentially important source of cells to treat neurodegenerative diseases. Our previous in vitro experiments showed that hNT cells possess immunocytochemically detectable markers typical of dopaminergic (DA) ventral mesencephalic (VM) neurons, including tyrosine hydroxylase (TH), dopamine transporter (DAT), dopamine receptor (D2), and aldehyde dehydrogenase (AHD-2). In the current study, we sought to examine whether Nurr1, an orphan receptor of the nuclear receptor superfamily shown to be essential for the development, differentiation and survival of midbrain DA neurons, would be expressed in 3, 4, or 5 week RA-induced hNT neurons and their NT2 precursors. Our immunocytochemical analyses indicate that NT2 cells as well as hNT neurons independent of the length of RA-driven differentiation were Nurr1-immunoreactive. RT-PCR analysis confirmed the expression of Nurr1-specific mRNA in both NT2 precursors and the hNT neurons. Furthermore, immunocytochemical co-expression of Nurr1 and TH was detected in hNT neurons. The findings of this study suggest that Nurr1 may be important during the development of hNT neurons and involved in their differentiation into the dopaminergic phenotype.     

Ascorbic acid responsive genes during neuronal differentiation of embryonic stem cells

Ascorbic acid has been reported to enhance differentiation of embryonic stem (ES) cells into neurons, however, the specific functions of ascorbic acid have not been defined yet. To address this issue, gene expression profiling was performed using cDNA microarray. Ascorbic acid increased the expressions of genes involved in neurogenesis, maturation, and neurotransmission. Furthermore, statistical analysis using Fisher's exact test revealed ascorbic acid significantly modulated the genes involved in cell adhesion and development category. These results provide information on the role for ascorbic acid during neuronal differentiation of ES cells and might contribute to large-scale generation of neurons for future clinical treatment.     

Neurogenesis in postnatal mouse dorsal root ganglia.

Neurogenesis continues in various regions of the central nervous system (CNS) throughout life. As the mitogen basic fibroblast growth factor (bFGF) can proliferate neuronal precursors of CNS neurons in culture, and is also upregulated within adult dorsal root ganglia following axotomy, it is possible that the postnatal dorsal root ganglia contain bFGF-responsive neuronal precursors. We undertook cell culture of postnatal mouse dorsal root ganglia to demonstrate neurogenesis. Basic FGF induced a cellular proliferative response in dorsal root ganglia cell culture. After 2 weeks in serum-free medium containing bFGF, neurons were rarely observed. However, following removal of bFGF and addition of trophic factors, many cells were observed that morphologically resembled dorsal root ganglia neurons, stained for neuronal markers, and generated action potentials. Furthermore, bromodeoxyuridine, used as a marker of cytogenesis, was detected in neurofilament-160(+) and/or microtubule-associated protein-2(+) cells that morphologically resembled neurons. In addition to bFGF, epidermal growth factor, nerve growth factor, and sonic hedgehog were also capable of generating spherical cell clusters that contained cells that stained for neuronal markers following the addition of trophic factors. These results suggest that early postnatal dorsal root ganglia contain neural precursors that appear to proliferate in response to various factors and can then be induced to differentiate into neurons. In conclusion, the existence of neural precursors and the possibility of neurogenesis in postnatal dorsal root ganglia may provide a greater range of plasticity available to somatosensory systems during growth or following injury, perhaps to replace ineffectual or dying neurons.     

Microarray analysis of fluoro-gold labeled rat dopamine neurons harvested by laser capture microdissection

The cellular heterogeneity of brain tissue presents a challenge to gene expression profiling of specific neuronal cell types. The present study employed a fluorescent neural tracer to specifically label midbrain dopamine neurons and non-dopamine cortical neurons. The labeled cells were then used to visually guide harvesting of the cells by laser capture microdissection (LCM). RNA extracted from the two populations of harvested cells was then amplified, labeled and co-hybridized to high density cDNA microarrays for two-color differential expression profiling. Many of the genes most highly enriched in the dopamine neurons were found to be genes previously known to define the dopamine neuronal phenotype. However, results from the microarray were only partially validated by quantitative RT-PCR analysis. The results indicate that LCM harvesting of specific neuronal phenotypes can be effectively guided in a complex cellular environment by specific pre-labeling of the target cell populations and underlie the importance of independent validation of microarray results.     

Impact of basic FGF expression in astrocytes on dopamine neuron synaptic function and development

Behavioural sensitization to amphetamine (AMPH) requires action of the drug in the ventral midbrain where dopamine (DA) neurons are located. In vivo studies suggest that AMPH sensitization requires enhanced expression of basic fibroblast growth factor (bFGF) in the nucleus of midbrain astrocytes. One idea is that the AMPH-induced increase in bFGF expression in astrocytes leads to enhanced secretion of this peptide and to long-term plasticity in DA neurons. To study directly the effects of astrocytic expression of bFGF on DA neurons, we established a cell-culture model of mesencephalic astrocytes and DA neurons. Immunolabelling showed that even in the absence of a pharmacological stimulus, the majority of mesencephalic astrocytes in culture express bFGF at a nuclear level. Arguing against the idea that bFGF was secreted, bFGF was undetectable in the extracellular medium (below 10 pg/mL). However, supplementing culture medium with exogenous bFGF at standard concentrations (20 ng/mL) led to a dramatic change in the morphology of astrocytes, increased spontaneous DA release, and inhibited synapse formation by individual DA neurons. RNA interference (siRNA) against bFGF mRNA, caused a reduction in DA release but produced no change in synaptic development. Together these data demonstrate that under basal conditions (in the absence of a pharmacological stimulus such as amphetamine) bFGF is not secreted even though there is abundant nuclear expression in astrocytes. The effects of bFGF seen here on DA neurons are thus likely to be mediated through more indirect glial-neuronal interactions, leading to enhanced DA release without a necessary change in synapse number.     

The neurotrophic effects of fibroblast growth factors on dopaminergic neurons in vitro are mediated by mesencephalic glia

Neurotrophic support is generally believed to result from a direct action of growth factors on developing neurons. However, there is increasing evidence that growth factors can indirectly affect neuronal development by glial-mediated processes. To investigate a possible role of glia in mediating neurotrophic effects on dopaminergic neurons, four purified growth factors were screened for dual effects on the survival and differentiation of dopaminergic neurons and on the proliferation of mesencephalic glial cells in vitro. Dissociated embryonic day 14.5 rat mesencephalon was grown at low cell density without serum, conditions under which both glial growth and neuronal survival are not optimal. Treatment of these cultures with acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) increased the number of surviving tyrosine hydroxylase-immunoreactive (TH-IR) neurons by 90-110% [corrected] at 8 d in vitro in a dose-dependent manner. The effects of these factors were not additive. High-affinity dopamine uptake was increased by bFGF, but not by aFGF. Length of TH-IR neurites was not affected by either aFGF or bFGF. Both growth factors induced proliferation of mesencephalic astrocytes as demonstrated by autoradiographic labeling with 3H-thymidine combined with immunocytochemistry for glial fibrillary acidic protein (GFAP). In contrast, platelet-derived growth factor (PDGF) and interleukin-1 had no effect on the survival or differentiation of dopaminergic neurons or the proliferation of mesencephalic astrocytes. Inhibition of glial proliferation abolished the neurotrophic effects exerted by aFGF or bFGF on dopaminergic neurons. Moreover, conditioned medium derived from mesencephalic glial cultures replated in the virtual absence of neurons also contained neurotrophic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse epidermal growth factor-responsive neural precursor cells increase the survival and functional capacity of embryonic rat dopamine neurons in vitro.

We have grown expanded populations of epidermal growth factor (EGF)-responsive mouse striatal precursor cells and subsequently co-cultured these with primary E14 rat ventral mesencephalon. The aim of these experiments was to induce dopaminergic (DA) neuronal phenotypes from the murine precursors. While no precursor cell-derived neurons were induced to express tyrosine hydroxylase (TH), there was a dramatic 30-fold increase in the survival of rat-derived TH-positive neurons in the co-cultures. The effect was not explicable solely in terms of total plating density, and was accompanied by a significantly enhanced capacity for [3H]dopamine uptake in the co-cultures compared to rat alone cultures. The present data show that, although primary rat E14 mesencephalic cells are incapable of inducing the development of DA neurons from EGF-responsive mouse neural precursor cells, such precursors will differentiate into cells capable of enhancing the survival and overall functional efficacy of primary embryonic dopamine neurons.     

Fibroblast growth factor stimulates photoreceptor differentiation in vitro.

Dissociated newborn rat retinal cells were maintained in monolayer culture for periods of up to 11 d. When grown in the absence of exogenous growth factors, 1-2% of the total neuronal population expressed opsin (the photopigment that is specific for maturing photoreceptors). Addition of a single dose of 10 ng/ml basic fibroblast growth factor (bFGF) to the culture medium induced an average increase of sixfold in the numbers of neurons expressing opsin. This supplementation had little effect on the total number of differentiated neurons or of glial cells when measured at the same time points. Furthermore, another specific class of retinal neurons, the amacrine cells, showed no changes following exposure to this growth factor. Two other growth factors known to exert neurotrophic effects, epidermal and nerve growth factor, were without effect. The effect of bFGF was dose dependent, with highly significant differences being observed with as little as 100 pg/ml, and with 700 pg/ml eliciting half-maximal stimulation; maximal effects were observed at 10 ng/ml. Induction of opsin expression by low concentrations of bFGF was blocked completely by an antiserum directed specifically against bFGF, but not by preimmune serum immunoglobulins. This increase in the number of photoreceptors expressing opsin following exposure to bFGF could have been due to either increased cell survival, increased proliferation of progenitor cells, or increased differentiation of immature photoreceptors. There was no increase in overall cell survival under the experimental conditions used, and double labeling immunocytochemistry combined with autoradiographic analysis of 3H-thymidine uptake showed that proliferation of neuronal precursors was not enhanced by the addition of bFGF. In contrast to these observations, cultures established from older (postnatal day 3) retina revealed large numbers of opsin-expressing photoreceptors in all culture plates, with or without added growth factors. This reduction in the stimulatory effects of bFGF with increasing postnatal age is consistent with the period of sensitivity being limited to the cycling of neuronal precursors. It is possible that a bFGF-like molecule is secreted by neighboring cells such as the retinal pigmented epithelium, to participate in retinal development and differentiation. To our understanding, this molecule is the first protein identified to influence specifically the differentiation of photoreceptor cells.     

Dopaminergic properties and function after grafting of attached neural precursor cultures

Generation of dopaminergic (DA) neurons from multipotent embryonic progenitors represents a promising therapeutical strategy for Parkinson's disease (PD). Aim of the present study was the establishment of enhanced cell culture conditions, which optimize the use of midbrain progenitor cells in animal models of PD. In addition, the progenitor cells were characterized during expansion and differentiation according to morphological and electrophysiological criteria and compared to primary tissue. Here, we report that CNS precursors can be expanded in vitro up to 40-fold and afterwards be efficiently differentiated into DA neurons. After 4-5 days under differentiation conditions, more than 70% of the neurons were TH+, equivalent to 30% of the total cell population. Calcium imaging revealed the presence of calcium-permeable AMPA receptors in the differentiated precursors which are capable to contribute to many developmental processes. The overall survival rate, degree of reinnervation and the behavioral performance after transplantation of 4 days in-vitro-differentiated cells were similar to results after direct grafting of E14 ventral mesencephalic cells, whereas after shorter or longer differentiation periods, respectively, less effects were achieved. Compared to the amount of in-vitro-generated DA neurons, the survival rate was only 0.8%, indicating that these cells are very vulnerable. Our results suggest that expanded and differentiated DA precursors from attached cultures can survive microtransplantation and integrate within the striatum in terms of behavioral recovery. However, there is only a short time window during in vitro differentiation, in which enough cells are already differentiated towards a DA phenotype and simultaneously not too mature for implantation. However, additional factors and/or genetical manipulation of these expanded progenitors will be required to increase their in vivo survival in order to improve both the ethical and the technical outlook for the use of fetal tissue in clinical transplantation.     

Methylphenidate exerts no neurotoxic, but neuroprotective effects in vitro

Summary. Methylphenidate (MPH) is the most common used drug in child and adolescent psychiatry. Despite of this fact, however, little is known about its exact pharmacological mechanisms. Here we investigated the toxic effects of MPH in vitro in human embryonic kidney (HEK-293) cells stably expressing the human dopamine transporter (HEK-hDAT cells) and in cultured rat embryonic (E14.5) mesencephalic cultures. MPH alone (up to 1 mM) affected neither the growth of HEK-hDAT cells nor the survival of dopaminergic (DA) neurons in primary cultures after treatment up to 72 h. No differences in neuronal arborisation or in the density of synapses were detected. 1-methyl-4-phenylpyridinium (MPP⁺) showed no toxic effect in HEK-293 cells, but had significant toxic effects in HEK-hDAT cells and DA neurons. MPH (1 μM – 1 mM) dose-dependently reduced this cytotoxicity in HEK-hDAT cells and primary mesencephalic DA neurons. The presented results show that application of MPH alone does not have any toxic effect on DA cells in vitro. The neurotoxic effects of MPP⁺ could be significantly reduced by co-application of MPH, an effect that is most likely explained by MPH blocking the DAT. The first and second authors contributed equally to this work     

Isolation and transplantation of dopaminergic neurons and neural stem cells

Although transplantation of mesencephalic tissue is considered a promising therapy for Parkinson's disease (PD), its clinical use is still restricted to a very few cases. A major limiting factor of this therapy is the difficulty of obtaining sufficient quantities of viable embryonic mesencephalic tissue. To overcome this limitation, techniques to produce dopaminergic (DA) neurons in vitro have been developed. However, these cultures are likely to contain a variety of unidentified cells, which must be removed before implantation. Specific cell-surface markers to sort DA neurons or their precursors are not available. We have developed an alternative strategy, by which these cells can be labeled with green fluorescent protein and isolated with fluorescent activated cell sorter. Transplantation of the sorted cells resulted in recovery of a rat model of the PD. This strategy should be useful for developing new therapies for PD.     

Loss of RE-1 silencing factor in mesenchymal stem cell-derived dopamine progenitors induces functional maturity

Stem cell-derived dopamine (DA) neurons hold great promise for Parkinson's disease (PD). Mesenchymal stem cells (MSCs) have great potential for clinical applications. The generation of DA cells from MSCs using sonic hedgehog (SHH) and fibroblast growth factors (FGF8 and bFGF) has been reported. However, the DA cells showed weak electrical properties, representing DA neuron progenitors. Since RE-1 Silencing Factor (REST), suppresses mature neuronal genes in neuronal progenitors, we studied its role in the maturation of MSC-derived DA cells. REST expression did not change during the induction process, thus we knocked down REST and subjected MSCs to the same neural induction cocktail. We observed increases in the protein level of the Na(+) voltage-gated channel and tyrosine hydroxylase (TH). Electrophysiological analyses showed spontaneous firings and spontaneous postsynaptic currents, similar to native DA neurons. Taken together, these results show REST as the limiting gene in the generation of functional mature neurons from MSCs.