Induction of a dopaminergic phenotype in cultured striatal neurons by bone morphogenetic proteins.

In the present study, we examined whether the bone morphogenetic proteins (BMPs), which are important in the developmental specification of transmitter type in certain classes of neurons, might also play a role in signaling the differentiation of a dopaminergic (DA) phenotype. We found that BMP-2, -4 and -6 were each capable of inducing, in a dose and time dependent manner, moderate levels of the DA enzyme tyrosine hydroxylase (TH) in cultured neurons from the mouse embryonic striatum. In contradistinction to other TH-inducing agents, BMPs initiated de novo TH expression without the required synergy of exogenous growth factors or co-activating substances and in neurons presumably aged (E16) beyond the critical period for induction. However, the appearance of TH in induced cells was short-lived (24 h) and could not be prolonged by repeated supplementation with the BMPs. Inhibitors of the mitogen-activated protein kinase (MAPK/ERK) signaling pathway, PD98059 and apigenin, did not prevent TH induction by BMP-4, as they did other TH inducing agents, indicating that the MAPK/ERK pathway does not mediate BMPs effects on TH expression. We conclude that BMP-2, -4 and -6 can be added to the expanding inventory of agents capable of inducing TH, making them potentially important in the specification of a DA phenotype in stem/precursor cells for the treatment of Parkinson's disease.

Differentiation of human dopamine neurons from an embryonic carcinomal stem cell line.

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine, TPA, IBMX/forskolin), will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study, we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media. Moreover, the induced TH present in these cells was enzymatically active, resulting in the production of low levels of dopamine (DA) and its metabolite DOPAC. These findings suggest that hNTs may provide an important tissue culture model for the study of factors regulating TH gene expression in human neurons. Moreover, hNTs may serve, in vivo, as a source of human DA neurons for use in transplantation therapies.

Sonic hedgehog and FGF8: inadequate signals for the differentiation of a dopamine phenotype in mouse and human neurons in culture.

Embryonic mouse striatal neurons and human neurons derived from the NT2/hNT stem cell line can be induced, in culture, to express the dopaminergic (DA) biosynthetic enzyme tyrosine hydroxylase (TH). The novel expression of TH in these cells is signaled by the synergistic interaction of factors present in the media, such as fibroblast growth factor 1 (FGF1) and one of several possible coactivators [DA, phorbol 12-myristate 13-acetate (TPA), isobutylmethylxanthine (IBMX), or forskolin]. Similarly, in vivo, it has recently been reported that the expression of TH in the developing midbrain is mediated by the synergy of FGF8 and the patterning molecule sonic hedgehog (Shh). In the present study, we examined whether the putative in vivo DA differentiation factors can similarly signal TH in our in vitro cell systems. We found that FGF8 and Shh induced TH expression in fewer than 2% of NT2/hNT cells and less than 5% of striatal neurons. The latter could be amplified to as much as 30% by increasing the concentration of growth factor 10-fold or by the addition of other competent coactivators (IBMX/forskolin, TPA, and DA). Additivity/inhibitor experiments indicated that FGF8 worked through traditional tyrosine kinase-initiated MAP/MEK signaling pathways. However, the Shh signal transduction cascade remained unclear. These data suggest that cues effective in vivo may be less successful in promoting the differentiation of a DA phenotype in mouse and human neurons in culture. Thus, our ability to generate DA neurons from different cell lines, for use in the treatment of Parkinson's disease, will depend on the identification of appropriate differentiation signals for each cell type under investigation.

Neurotransmitters, KCl and antioxidants rescue striatal neurons from apoptotic cell death in culture.

Striatal neurons grown in low density culture on serum-free media and in the absence of glia die within 3 days of plating. In this study, we sought to determine the mechanism of cell death (e.g., apoptosis) and whether trophic influences, such as, growth factors, neurotransmitters, antioxidants or KCl-mediated depolarization could improve their survival. We found that striatal neurons grown in this manner die via apoptosis unless treated with one of several different rescuing agents. One way to prevent the death of most striatal neurons was continual treatment with 5-20 microM dopamine (DA) or other monoamines. Although the survival effect of DA was mimicked by the specific D1 receptor agonist, SKF38393, no D1 or D2 receptor antagonists blocked the effect. As with DA, chronic depolarization with KCl (12-39 mM) or treatment with antioxidants, such as the vitamin E analog, Trolox (10-10-500 microM), or the hormone, melatonin (10-10-500 microM) also rescued striatal neurons from impending cell death. Surprisingly, growth factors, such as BDNF, bFGF, GDNF, NGF, NT3 and EGF, demonstrated no ability to rescue striatal neurons in this model, suggesting that death was not solely caused by the absence of essential trophic factors. We conclude that a variety of agents, but not growth factors, can prevent the demise of striatal neurons, presumably by neutralizing damage at one or more steps in the death cascade.

Melatonin rescues dopamine neurons from cell death in tissue culture models of oxidative stress.

Dopamine (DA) neurons are uniquely vulnerable to damage and disease. Their loss in humans is associated with diseases of the aged, most notably, Parkinson's Disease (PD). There is now a great deal of evidence to suggest that the destruction of DA neurons in PD involves the accumulation of harmful oxygen free radicals. Since the antioxidant hormone, melatonin, is one of the most potent endogenous scavengers of these toxic radicals, we tested its ability to rescue DA neurons from damage/death in several laboratory models associated with oxidative stress. In the first model, cells were grown in low density on serum-free media. Under these conditions, nearly all cells died, presumably due to the lack of essential growth factors. Treatment with 250 microM melatonin rescued nearly all dying cells (100% tau+ neurons), including tyrosine hydroxylase immunopositive DA neurons, for at least 7 days following growth factor deprivation. This effect was dose and time dependent and was mimicked by other antioxidants such as 2-iodomelatonin and vitamin E. Similarly, in the second model of oxidative stress, 250 microM melatonn produced a near total recovery from the usual 50% loss of DA neurons caused by neurotoxic injury from 2.5 microM 1-methyl-4-phenylpyridine (MPP+). These results indicate that melatonin possesses the remarkable ability to rescue DA neurons from cell death in several experimental paradigms associated with oxidative stress.

Expression of tyrosine hydroxylase in newly differentiated neurons from a human cell line (hNT).

Previous studies have demonstrated that the synergistic interaction of acidic fibroblast growth factor (aFGF) and a number of co-activator molecules (dopamine, TPA, IBMX/forskolin) can induce the novel expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in non-TH-expressing neurons. To date, TH gene induction has been achieved only in cultures of primary brain neurons. In the present study, we investigated whether TH expression could similarly be induced in a cell line derived from human teratocarcinoma cells. Treatment with aFGF and its co-activators resulted in the prolonged expression of TH in newly differentiating human neurons (hNT) but not in their undifferentiated precursors (NT2). These findings suggest that hNTs may serve as a continual source of TH-expressing neurons for cell transplantation and developmental studies.

Acidic fibroblast growth factor and catecholamines synergistically up-regulate tyrosine hydroxylase activity in developing and damaged dopamine neurons in culture.

Our previous studies indicate that, in certain non-catecholamine (CA) neurons, expression of the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH) can be initiated by the obligatory interaction of acidic fibroblast growth factor (aFGF) and a CA activator. In this study, we sought to determine whether these same differentiation factors also play a role in regulating existing TH expression in CA neurons. Thus, the effects of exogenous aFGF and CAs on TH were studied in developing or toxin-damaged dopamine (DA) neurons from the embryonic day 15 rat ventral midbrain, where it was likely to be at physiologically low levels. Cultures were incubated with various concentrations of aFGF, DA, or aFGF and DA. Some cultures were first damaged with 2.5 microM 1-methyl-4-phenylpyridinium. In developing DA neurons, an 80% increase in TH activity was found only after co-treatment with aFGF (100 ng/ml) and DA (1 microM) or other monoamines. Likewise, in damaged DA neurons, aFGF and DA reversed the 50% loss in TH activity caused by toxin. This was observed within 4 h of treatment and was not associated with changes in the number or appearance of DA neurons, suggesting a biochemical rather than a trophic effect. Pretreatment with protein or RNA synthesis inhibitors eliminated the increase. In PC12 cells, where TH is highly expressed, activity was unaltered by treatment. We conclude that the aFGF and CAs may be involved in not only the initiation but also the regulation of TH.

Brain-derived neurotrophic factor works coordinately with partner molecules to initiate tyrosine hydroxylase expression in striatal neurons.

Previous studies demonstrated that the cooperative interaction of acidic fibroblast growth factor (aFGF) and a partner molecule could induce the novel expression of the catecholamine (CA) biosynthetic enzyme, tyrosine hydroxylase (TH) in striatal neurons [Du and Iacovitti, J. Neurosci., in press; Du et al., J. Neurosci., 14 (1994) 7688-7694; Iacovitti et al., submitted]. The present study demonstrates that in addition to aFGF, brain-derived neurotrophic factor (BDNF) is also capable of moderate levels of TH induction (30% TH+ striatal neurons) when administered at high concentrations (100 ng/ml). As with aFGF, BDNF's activity depended on its coupling to an appropriate partner molecule; the most potent of which were 10 microM dopamine (DA) and 50 microM mazindol. BDNF + DA-induced TH expression was first evident after at 12 h; peaked by 18 h and declined by 4 days in culture. Cyclohexamide eliminated nearly all and alpha-amanitin reduced by half the TH induction elicited by DA and BDNF; indicating that both de novo transcription and translation were required for increased expression. In contrast with aFGF and BDNF, other putative dopamine differentiation factors, such as glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), were able to elicit barely detectable (10%) levels of TH induction, regardless of the partner molecule used. These studies suggest that aFGF and/or BDNF may work coordinately with partner molecules to initiate TH expression; while a number of factors including, CNTF and GDNF, may be involved in its subsequent modulation.

Purification of dopamine neurons by flow cytometry.

The heterogeneity and preponderence of other cell types present in cultures has greatly impeded our ability to study dopamine neurons. In this report, we describe methods for isolating nearly pure dopamine neurons for study in culture. To do so, the lipid-soluble dye, 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (diI) was injected into the embryonic rat striata where it was taken up by nerve terminals and transported overnight back to the innervating perikarya in the ventral midbrain. Midbrain cells were then dissected, dissociated and separated on the basis of their (rhodamine) fluorescence by flow cytometry. Nearly all cells recovered as fluorescent positive (> 98%) were also immunoreactive for the dopamine specific enzyme tyrosine hydroxylase (80%-96%). Little contamination by other cells types was observed after labeling for specific neuronal and glial markers. Purified dopamine neurons continued to thrive and elaborate neuronal processes for at least 3 days in culture. Using this new model, it may now be possible to directly study the cellular and molecular processes regulating the survival and functioning of developing, injured and transplanted dopamine neurons.

Novel expression of the tyrosine hydroxylase gene requires both acidic fibroblast growth factor and an activator.

Substances found in the soluble extract of muscle can alter the differentiative fate of certain brain neurons in culture by triggering novel expression of the gene for the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) (Iacovitti et al., 1989; Iacovitt, 1991). In this study, we demonstrate that TH induction in cultured noncatecholamine neurons from the mouse striatum requires the cooperative interaction of at least two substances found in muscle. Purification studies, combined with biological assay, revealed that one necessary component is acidic fibroblast growth factor (aFGF), and the other, an unidentified molecule(s) of < 10 kDa molecular weight that activated aFGF. Thus, muscle-derived aFGF, if incubated in the presence but not the absence of the < 10 kDa fraction of muscle, induced a dose-dependent increase in the number of striatal neurons that novelly express TH. This expression was blocked by prior incubation and protein A precipitation of the factor with polyclonal antibodies to aFGF (1:200-1:1000). Similar to muscle-purified aFGF, commercial preparations of native bovine and human recombinant aFGF (0.1-100 ng/ml) were potent inducers of TH when coincubated with the < 10 kDa activator. In contrast, basic FGF produced little and FGF-7 no induction of TH. Unlike the unidentified activating agent in muscle, heparin (20-500 mU), a known activator of aFGF, did not potentiate the factor's TH-inducing activity. Nonetheless, heparatinase (100 mU) prevented TH induction by aFGF and its activator, indicating that binding of heparan sulfated proteoglycans is necessary for the effect.(ABSTRACT TRUNCATED AT 250 WORDS)

GM1 ganglioside partially rescues cultured dopaminergic neurons from MPP(+)-induced damage: dependence on initial damage and time of treatment.

GM1 ganglioside is believed to be important in promoting the recovery of neurons from injury. The present study assesses the ability of GM1 to repair or prevent the damage of dopamine neurons caused by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Treatment of mesencephalic cell cultures with 2.5 microM MPP+ resulted in the loss of 30% of tyrosine hydroxylase (TH) immunoreactive neurons. In contrast, cultures administered 100 microM GM1 ganglioside for 3 days after toxin treatment contained nearly control numbers of TH+ neurons (97%). This reparative effect of GM1 was reflected in parallel increases in TH enzyme activity, dopamine and dopac levels. Cultures sustaining greater insult from higher doses of MPP+ (5.0-10.0 microM) did not benefit from ganglioside treatment, suggesting that rescue by GM1 depended on the degree of initial damage to cells. Moreover, the timing of ganglioside treatment was critical; pretreatment with GM1 alone did not prevent or attenuate the damage caused by subsequent incubation in 2.5 microM MPP+.

Muscle-derived differentiation factor increases expression of the tyrosine hydroxylase gene and enzyme activity in cultured dopamine neurons from the rat midbrain.

Our earlier work demonstrated that certain populations of brain neurons which do not synthesize catecholamine (CA) neurotransmitters in vivo, will, when grown in culture with muscle-derived differentiation factor (MDF), unexpectedly express the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH). In this paper, we sought to determine whether MDF could also regulate TH expression in those neurons which normally synthesize CA neurotransmitters. Incubation of cultured dopamine neurons from the ventral midbrain with MDF elevated the levels of TH mRNA and TH enzyme activity 5- to 40-fold higher than that measured in control cultures. Sympathetic neurons were unaffected by a similar MDF treatment. Unlike the 2-day critical period for MDF-responsivity in non-CA neurons. CA neurons remained susceptible to MDF's influence over an extended developmental interval (E14-18), suggesting that MDF may be important for TH gene regulation in brain CA neurons even differentiation is complete. Because of these unique properties, MDF may provide a unique opportunity to explore ways in which the TH gene might be directly manipulated in these cell populations in order to correct the CA imbalances that occur in certain neurological diseases and disorders.