Risultati finali
Dopaminergic neurons are located in many regions in the CNS and are characterized by the expression of tyrosine hydroxylase (TH). TH catalyzes the rate-limiting step in the biosynthesis of dopamine utilizing tyrosine, molecular oxygen and tetrahydrobiopterin as co substrates in the formation of 3,4-dihydroxyphenylalanine. Dopaminergic neurons derived from the ventral midbrain are of special interest because of the selective loss of this cell population in patients with Parkinsons disease (PD).
However, the study of this population of cells has been difficult because of the heterogeneity of cell cultures established from the midbrain. In most instances, dopaminergic neurons comprise five percent or less of the total cell population, which also complicates drug screening and gene discovery based on such cultures. The establishment of a method to purify dopaminergic neurons will be beneficial in the context of implanting developing dopaminergic neurons originating from aborted human fetuses in the brains of patients with PD (Bjorklund, Novartis Found Symp 2000; 231:7-15).
Although a successful restoration of function in the patients was observed in many cases, undesirable side-effects were observed in a recent study (Freed et al., 2001 N. Engl. J. Med. 344, 710-9). The problems in this study may be caused by the use of heterogeneous cell populations and uncertainties in number of dopaminergic cells transplanted (Dunnett; Nat Rev Neurosci 2001 May; 2 (5): 365-9).
Dopaminergic neurons can be visualized in formalin-fixed cell preparations, by immunostaining for tyrosine hydroxylase (TH). However, presently a method to quantify and isolate this specific cell population without harming the cells remains to be established. We show in the present project that antibodies to Fetal antigen 1 (FA1) can be used to isolate dopaminergic neurons.
FA1 is one of the increasing numbers of proteins belonging to the epidermal growth factor (EGF)-superfamily that have been identified within the last decade. FA1 is synthesized as a larger transmembrane precursor and released from cells after proteolytic action of an unidentified enzyme. Several groups have described cDNA clones for this precursor, each assigning a new name for the cDNA depending on the species and tissue/cell type from which they isolated it.
Expression of FA1 in the adult CNS has been observed (Harken-Jensen Ph.D. thesis; Odense Univ. 1999) and FA1 expression has been observed in the fetal CNS (Floridon et al., Differentiation 2000 66(1), 49-59) both authors gave preliminary results wherein the cellular location of expression was not determined. Together with our collaborators at Odense University (Harken-Jensen and Teisner), we have in this study demonstrated that the FA1 expression is a hallmark of dopaminergic neurons.
We show in the present study that FA1 antibody can be used to separate dopaminergic neurons or neural stem/progenitor cells from other neural cells. Generally, a cell suspension prepared from human CNS tissue (e.g. from human fetal brain) is brought into contact with a FA1 antibody and cells that bind FA1 antibody are then separated from cells not expressing FA1. The CNS tissue may be taken from any part of the brain or spinal cord and may be selected by dissection of particular regions, which contain particular cell types. For instance the ventral mesencephalon may be selected to provide dopaminergic neurons and the substantia nigra pars compacta is particularly rich in dopaminergic neurons.
Cell sorting using FA1 antibodies may be useful for isolation of a highly enriched source of dopaminergic neurons for transplantation into patients with PD. Antibodies that label the populations of neural stem cells, neural progenitor cells and their differentiated progeny are extremely useful in drug screening, gene discovery and for transplantation purposes because they allow the enrichment of populations of e.g. dopaminergic neurons or their progenitors in a single step. Cells recovered with FA1 antibody derived from different stages in their development could be used in studies on the mechanisms of action of cells, factors, and genes that regulate dopaminergic cell proliferation and differentiation. Furthermore, dopaminergic neurons from normal and pathological brain tissue may be recovered using FA1 antibodies and compared.
Two mouse embryonic cell lines were generated that have the potential to express the Green Fluorescent Protein GFP in a conditional manner. This means that using the Cre recombinase system of the phage P1, a specific cell type can be labelled with GFP, when a specific promoter for that cell type is provided to control Cre expression. The GFP was fused to the membrane portion of the GAP-34 protein, so that it is targeted to the cell membrane. From these two cell lines two transgenic mouse lines were also generated. These transgenic mouse lines allow also the conditional expression of GFP in living animals and provide a useful tool to label specific cell types or organs, when a specific promoter is available to allow Cre recombinase specific expression. For this purpose, our mouse lines should be crossed to transgenic mice that express the Cre recombinase under the desired specific promoter.
In neural transplantation studies there is an interest in identifying and isolating mesencephalic dopamine (mesDA) neuron precursors that have the capacity to differentiate into fully mature mesDA neurons after transplantation. We report here that in the developing ventral mesencephalon (VM) the proneural gene Neurogenin2 (Ngn2) is expressed exclusively in the part of the ventricular zone that gives rise to the migrating mesDA neuroblasts, but not in the differentiated mesDA neurons. Previous studies have demonstrated that Ngn2 is involved in induction of neurogenesis as well as in the specification of progenitor cell identity.
We show that cells isolated by FACS from the developing VM of Ngn2-GFP knock-in mice are capable of generating mesDA neurons, both in vitro and after transplantation to the striatum of neonatal rats. All mesDA neuron precursors, but not the serotonergic or GABAergic neuron precursors, are contained in the Ngn2-GFP-expressing population, indicating that Ngn2 is selectively involved in the generation of mesDA neurons within the developing VM. Furthermore, we report that surviving mesDA neurons in VM grafts are derived from early post-mitotic, probably Nurr1-expressing precursors before they have acquired their fully differentiated neuronal phenotype.
These results open possibilities to use expression of the proneural gene Ngn2 as a marker for an early progenitor of mesencephalic dopamine neurons. Nevertheless, no immediate use or commersial application is envisoned at this stage.
A complete account of the research finding is now in pree in Experimental Neurology.
The Wnts are a family of glycoproteins that regulate cell proliferation, fate decisions and differentiation. In our study we examined the contribution of Wnts to the development of ventral midbrain (VM) dopaminergic (DA) neurons. Our results show that b-catenin is expressed in DA precursor cells and that b-catenin signaling takes place in these cells, as assessed in TOPGAL reporter mice. We also found that Wnt-1, -3a and -5a are differentially regulated and that partially purified Wnts distinctively regulate VM development. Wnt-3a promoted the proliferation of Nurr1+ precursor cells but did not increase the number of TH+ neurons. Instead, Wnt-1 and -5a increased the number of rat midbrain DA neurons in rat E14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of Nurr1+ precursors, upregulated cyclin D1 and D3, and downregulated p27 and p57 mRNAs. In contrast, Wnt-5a primarily increased the proportion of Nurr1+ precursors that acquired a neuronal DA phenotype, which included the upregulation of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled 8 (a Wnt inhibitor) blocked endogenous Wnts and the effects of Wnt-1 and Wnt-5a on proliferation and the acquisition of a DA phenotype in precursor cultures. These findings indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and that different Wnts have specific and unique activity profiles.
We next explored the mechanism by which Wnt-5a promoted DA neuron differentiation and found that purified Wnt-5a protein induces the hyperphosphorylation of dishevelled but not the stabilization of b-catenin. Surprisingly however, we also found that stabilization of b-catenin after overexpression or treatment with GSK3beta inhibitors such as Indirubin 3 monoxime or kempaullone also results in the differentiation of Nurr1+ precursors into TH+ dopaminergic neurons. Thus, our results suggest that the differentiation of dopaminergic precursors into neurons can be enhanced by activation of both canonical and non-canonical Wnt pathways. Finally, we have recently found that Wnt-5a is secreted mainly by glial cells in the ventral midbrain and is part of the dopaminergic inductive activity derived from the glia. Moreover, we found that Wnt-5a blocking antibodies block the effects of ventral midbrain glia on the differentiation of neurospheres into dopaminergic neurons, indicating that Wnt-5a is in part required for the dopaminergic differentiation of stem cells. Thus, our results suggest that Wnt-5a could be applied to promote the differentiation of stem cells into dopaminergic neurons.
The therapeutic potential of fetal VM neuron transplantation in patients with Parkinson¡¦s disease (PD) has been demonstrated in the clinic. Immature dopamine (DA) neurons originating from aborted human fetuses, implanted in the brains of patients with end stage PD, have successfully restored function (Lindvall, O. et al, Progress in Brain Research 2000, 127: 299-320.). Despite the positive proof-of-principle, neural transplantation has not become widely applied, primarily due to the limited availability and ethical concerns of the use of fetal VM tissue. In addition, poor survival and heterogeneous and poorly characterized primary cell materials have made the analysis of experimental data difficult, and results and complications have varied greatly between different transplantation centers (Brundin, P. et al, Brain, 2000, 123: 1380-90.3, Freed, C. R. et al, New England Journal of Medicine 2001, 344: 710-9. Piccini, P. et al, Nature Neuroscience 1999, 2: 1137-40). Therefore, there is currently an extensive search for alternative sources of transplantable cells capable of reproducing the positive results seen with primary fetal VM while being better characterized and less heterogeneous.
Multipotent stem/progenitor cells derived from human first trimester forebrain can be expanded as free-floating aggregates. In vitro, cells can differentiate to neurons, astrocytes and oligodendrocytes. Using previously described protocols for in vitro differentiation, a large fraction of the neurons become ×-aminobutyric acid (GABA)-immunoreactive, whereas only rare tyrosine hydroxylase (TH) expressing neurons are found. The present report describes conditions under which 4-10 % of the cells in the culture become immunoreactive (ir) for TH within 24 h. Factors including acidic Fibroblast Growth Factor (aFGF) in combination with agents that increase intracellular cyclic AMP and activate protein kinase C (PKC) in addition to a substrate that promotes neuronal differentiation appear critical for efficient TH induction. The cells remain THir after trypsinization and replating and in the absence of inducing factors. Consistent with a dopaminergic phenotype, mRNAs encoding aromatic acid decarboxylase (AADC) but not dopamine-Ò-hydroxylase (DBH) was detected by Q-PCR. Although, differentiated human neurons were demonstrated 10 weeks after implantation into the striatum of adult rats with unilateral dopaminergic lesions, only few THir neurons could be observed.
The method involves plating the cells on a substrate known to promote neuronal differentiation, preserving cell-cell interaction, and exposing the cells to a cocktail consisting of trophic factors and agents that affect intracellular signaling. The ability to induce TH expression is not developmentally dependent but appears to be a general phenomenon of human neurosphere cultures established from first trimester forebrain.
In the present project, we have shown that, under certain defined conditions, human forebrain neurosphere cultures can generate a significant number of neurons expressing dopaminergic neuronal markers in a similar fraction of cells as compared to fetal VM cultures (4-10 %). Based on these findings we have developed a method for the in vitro production of a population of neural cells wherein a significant percentage of those cells express tyrosine hydroxylase (TH). The relative importance of several parameters including substrate, growth factors, metabolic agents, and plating density and dissociation has been extensively studied to generate an optimized protocol. This protocol comprises expanding neural progenitor cells using growth factors and for by plating the cells on a substrate, introducing a defined culture medium containing one or more growth factors belonging to the FGF family, a molecule which gives to an increase in intracellular cyclic AMP (CAMP), and an agent stimulating or capable of activating protein kinase C (PKC). The method provides TH expressing cells in significant numbers, similar to that observed in fetal ventral mesencephalon cultures (5-20%). It also provides cells in which the expression of TH is stable after removal of the induction medium. The method provides a means for generating large numbers of TH expressing neural cells for neurotransplantation into a host in the treatment of CNS disorders, for example, neurodegenerative disease, neurological trauma, stroke, other neurodegenerative diseases, neurological trauma, stroke, and other diseases of the nervous system involving loss of neural cells, particularly Parkinson's disease. However, additional experimental work is required that will improve the maturation and survival of the TH expressing neurons after implantation in vivo before these cultures can become useful in a cell replacement therapy for PD. Additionally, the TH expressing cells may be used for drug screening or gene expression analysis.
Progress in stem cell biology research is enhancing our ability to generate specific neuron types for basic and applied studies and to design new treatments for neurodegenerative diseases. In the case of Parkinsons disease (PD), alternative human dopaminergic (DAergic) neurons other than primary fetal tissue do not yet exist. One possible source could behumanneural stem cells (hNSCs), although the yieldin DAergic neurons and their survival are very limited.
In this study, we found that Bcl-XL enhances (one-to-two orders of magnitude) the capacity for spontaneous dopaminergic differentiation of hNSCs, which then exceeds that of cultured human ventral mesencephalic tissue. Bcl-XL also enhanced total neuron generation by hNSCs, but to a lower extent. Neuronal phenotypes other than DA were not affected by Bcl-XL , indicating an exquisitely specific effect on DAergic neurons. In vivo, grafts of Bcl-XL-overexpressing hNSCs do generate surviving human TH neurons in the adult rat 6-OHdopamine lesioned striatum, something never seen when naive hNSCs were transplanted. Most of the data obtained here in terms of the effects of Bcl-XL are consistent with an enhanced survival type of mechanism and not supportive of induction, specification, or proliferation of DAergic precursors.
From this in vitro and in vivo evidence, we conclude that enhancing Bcl-XL expression is important to obtain human DAergic neurons from hNSCs. These findings may facilitate the development of drug-screening and cell-replacement activities to discover new therapeutic strategies for PD.
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