EP1827470A2 - Materials and methods related to dickkopfs (dkk) and neurogenesis - Google Patents

Abstract

Materials and Methods related to Dickkopfs and Neurogenesis Abstract Methods for promoting dopaminergic neuronal development and producing neural cells having a dopaminergic phenotype. Dopaminergic neural cells may be used for treating individuals having a neurodegenerative disease such as Parkinson's disease. Dopaminergic cells may be implanted into the brain of the individual, and/or dopaminergic neural development may be induced or enhanced in the brain of the individual. Methods comprise treating the cell with a Dkk ligand, such as any one of Dkk1 to Dkk4 or a fragment thereof comprising a cysteine rich domain, or a Dkk receptor such as LRP5 or LRP6, thereby producing or enhancing proliferation, self-renewal, survival and/or dopaminergic induction, differentiation, survival or acquisition of a neuronal dopaminergic phenotype. The cell may be co-cultured with astrocytes or glial cells and may be contacted with an FGF growth factor. Dopaminergic neurons may also be useful in drug/toxicology testing and for target development or drug discovery.

Description

Materials and Methods related to Dickkopfs and Neurogenesis

The present invention relates to induction of neuronal fate in neural stem cells or neural progenitor or precursor cells, or other stem cells. It relates to induction and enhancement of induction of a specific neuronal phenotype, and particularly to induction and enhancement of induction of a midbrain dopaminergic neuronal phenotype.

Parkinson's disease (PD) is a very common neurodegenerative disorder whose pathogenesis is characterized by a selective and progressive loss of midbrain dopaminergic (DA) neurons . The enhancement of induction of neuronal phenotype has the potential to allow for treatment of Parkinson's disease and other seriously debilitating neurodegenerative disorders.

Previously, human fetal mesencephalic tissue has been grafted into Parkinsonian patients with positive results, but development of specific cell replacement therapies utilizing the present invention overcomes practical and ethical difficulties with such prior approaches. In particular, the present invention allows for development of cell preparations for transplantation while reducing or eliminating any need for use of embryo tissue or embyronic cells. Stem cells may be obtained from the umbilical cord, a tissue that is normally discarded. Another option to is to obtain adult stem cells, e.g. from bone marrow, blood, skin, eye, olfactory bulb or olfactory epithelia.

The induction of specific neuronal phenotypes requires the integration of both genetic and epigenetic signals. In the developing midbrain, the induction of dopaminergic neurons has been reported to require the orphan nuclear receptor Nurrl (Zetterstrδm et al., 1997; Saucedo-Cardenas et al . , 1998; Castillo et al. , 1998), but expression of Nurrl is not sufficient to induce a dopaminergic phenotype in neural stem cells (Wagner et al . , 1999) . The findings set out herein indicate that Dickkopf (Dkk) ligands and receptors are key regulators of proliferation, self-renewal, differentiation and fate decisions during ventral midbrain neurogenesis. Moreover, our results pave the way for the large-scale production of midbrain DA neurons in vitro and for the future implementation of stem cell replacement strategies in the treatment of neurodegenerative diseases such as Parkinson's disease (Bjorklund and Lindvall 2000; Price and Williams 2001; Arenas, 2002 Rossi and Cattaneo, 2002; Gottlieb et al. , 2002) .

Embryonic, neural and multipotent stem cells have the ability to differentiate into neural cell lineages including neurons, astrocytes and oligodendrocytes. Moreover, stem cells can be isolated, expanded, and used as source material for brain transplants (Snyder, E. Y. et al. Cell 68, 33-51 (1992); Rosenthal, A. Neuron 20, 169-172 (1998); Bain et al. , 1995; Gage, F.H., et al. Ann. Rev. Neurosci. 18, 159-192 (1995); Okabe et al. , 1996; Weiss, S. et al. Trends Neurosci. 19, 387-393 (1996); Snyder, E. Y. et al. Clin. Neurosci. 3, 310-316 (1996); Martinez-Serrano, A. et al.

Trends Neurosci. 20, 530-538 (1997); McKay, R. Science 276, 66-71 (1997); Deacon et al . , 1998; Studer, L. et al . Nature Neurosci. 1, 290-295 (1998) ; Bjorklund and Lindvall 2000; Brustle et al . , 1999; Lee et al . , 2000; Shuldiner et al. , 2000 and 2001; Reubinoff et al . , 2000 and 2001; Tropepe et al . , 2001; Zhang et al., 2001; Price and Williams 2001; Arenas 2002; Bjorklund et al . , 2002; Rossi and Cattaneo, 2002; Gottlieb et al . , 2002) .

Most neurodegenerative diseases affect neuronal populations. Moreover, most of the damage occurs to cells with a specific neurochemical phenotype. In human Parkinson's disease, for example, the major cell type lost is midbrain dopaminergic neurons. Functional replacement of specific neuronal populations through transplantation of neural tissue represents an attractive therapeutic strategy for treating neurodegenerative diseases (Rosenthal, A. Neuron 20, 169-172 (1998); Arenas E., Brain Res Bull. 57 (6) :795-808 (2002) ; Lindvall O., Pharmacol Res. 47(4) :279-87 (2003) ; Bjδrklund A. et al. , Lancet Neurol. 2(7) :437-45 (2003) .) . Another alternative would be the direct infusion of signals required to promote regeneration, repair or guide the development and/or recruitment of stem or progenitor or precursor cells, or the administration of drugs that regulate those functions.

Stem/progenitor or precursor cells are an ideal material for transplantation therapy since they can be expanded and instructed to assume a specific neuronal phenotype. These cells would circumvent ethical and practical issues surrounding the use of human foetal tissue for transplantation.

Induction of a single and specific neuronal phenotype in stem or progenitor or precursor cells has proven elusive.

The present invention provides for induction of dopaminergic neuronal phenotype in cells. Thus, by regulating the levels of Dickkopf ligands such as Dickkopf-1, -2, -3 or -4 (Dkks) and/or Dkk receptors such as low density lipoprotein receptor (LDLR) -related protein-5 or -6 (LRP5/6) in cultures or in the brain, the invention allows the induction or promotion of: proliferation and/or self- renewal of dopaminergic precursors, progenitor or stem cells,- and/or promotion of dopaminergic neuron, precursor, progenitor or stem cell survival, differentiation and maturation, increasing the yield of dopaminergic neurons; and/or induction of a neuronal dopaminergic fate in stem, progenitor, precursor or neuronal cells in vitro or in vivo.

Any aspect or embodiment of the invention can apply to or use a stem, precursor, progenitor or neuronal cell i.e. a neuron. A 'neural cell' in the present disclosure may be a neuronal cell. Cell preparations rich in dopaminergic neurons may be used for cell replacement therapy in Parkinson's disease or other disorders, and for studying signalling events in dopaminergic neurons and the effects of drugs on dopaminergic neurons in vitro, for instance in high throughput screening.

Aspects and embodiments of the present invention are provided as set out in the claims below.

In one aspect, the present invention provides a method of inducing a dopaminergic neuronal fate in a stem cell, neural stem cell, embryonic stem cell or neural progenitor or precursor cell, or enhancing dopaminergic induction or differentiation in a neuronal cell, or expanding a dopaminergic precursor or progenitor cell, the method comprising: modulating the level and/or activity of a Dkk ligand/receptor polypeptide in said cell, whereby dopaminergic neurons are produced.

The invention provides a method of inducing or promoting dopaminergic neuronal development by enhancing proliferation, self- renewal, dopaminergic induction, survival, differentiation and/or maturation in a neural stem, progenitor or precursor cell, or other stem or neural cell, the method comprising: modulating the level and/or activity of a Dkk ligand/receptor polypeptide in said cell, thereby producing or enhancing proliferation, self-renewal, survival and/or dopaminergic induction, differentiation, neurotransmission, survival or acquisition of a neuronal dopaminergic phenotype.

A Dkk ligand/receptor polypeptide is a polypeptide able to form a Dkk receptor/ligand complex with a suitable binding partner. Dkk ligand/receptor polypeptides include polypeptides which are Dkk ligands and polypeptides which are Dkk receptors. Examples of Dkk ligand/receptor polypeptides include Dkk polypeptides, such as Dkk- 1, Dkk-2, Dkk-3 or Dkk-4, which bind Dkk receptors, and Dkk receptors, such as LRP5, LRP6, Kremen 1 and Kremen 2, which bind Dkk polypeptides. Dkk ligand/receptor polypeptides may be endogenous cellular polypeptides or may be synthetic polypeptides produced by recombinant or other means.

A Dkk receptor is a polypeptide which is able to form a Dkk receptor/ligand complex with a Dkk polypeptide. Suitable Dkk receptors include LRP5, LRP6, Kremen 1 and Kremen 2.

A Dkk ligand is a peptidyl or non-peptidyl molecule which is able to form a Dkk receptor/ligand complex with a Dkk receptor (i.e. a Dkk receptor agonist) . Examples of Dkk ligands include Dkk polypeptides as described below.

A Dkk polypeptide may be a Dkk-1, -2, -3 or -4 polypeptide from any mammalian species, for example a mouse Dkk-1, -2, -3 or -4 or a human Dkk-1, -2, -3 or -4, or a Dkk polypeptide may be a fragment or variant of any one of these. For example, a Dkk-1 polypeptide may comprise a proteolytic fragment as shown in figure 11 and a Dkk-2 polypeptide may comprise a proteolytic fragment as shown in figure 12.

A Dkk polypeptide may comprise or consist of one or both of the two cysteine rich domains (Cys-1 or Cys-2) of a wild-type Dkk sequence, such as human Dkkl, Dkk2, Dkk3 or Dkk4, or may comprise or consist of a variant of one or both of these domains .

The Cys-1 domain of human Dkkl consists of residues 85 to 138 of the full-length sequence and the Cys-2 domain of human Dkkl consists of residues 189 to 263 of the full-length sequence.

The Cys-1 domain of human Dkk2 consists of residues 78 to 127 of the full-length sequence and the Cys-2 domain of human Dkk2 consists of residues 183 to 256 of the full-length sequence. The Cys-1 domain of human Dkk3 consists of residues 147 to 195 of the full-length sequence and the Cys-2 domain of human Dkk3 consists of residues 208 to 284 of the full-length sequence.

The Cys-1 domain of human Dkk4 consists of residues 41 to 90 of the full-length sequence and the Cys-2 domain of human Dkk4 consists of residues 145 to 218 of the full-length sequence.

A fragment or variant of a wild-type Dkk sequence as described herein may differ from the wild-type sequence by the addition, deletion, substitution and/or insertion of one or more amino acids, provided the function of modulating development of a dopaminergic neuronal fate in a stem cell, neural stem cell, embryonic stem cell or neural progenitor or precursor cell is retained. (Monaghan et al. (1999) , Bafico et al . , (2001); Li et al . (2002) ; MacDonald et al. (2004); Mao et al . (2001a) ; Semenov et al. , (2001) .

For example, a polypeptide which is a variant of a wild-type sequence may comprise an amino acid sequence which shares greater than about 30% sequence identity with the wild-type sequence, for example human Dkk-1, -2, -3, or -4 or one or more domains thereof, greater than about 40%, greater than about 45%, greater than about 55%, greater than about 65%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%. The sequence may share greater than about 30% similarity with the wild- type sequence, for example human Dkk-1, -2, -3, or -4 or one or more domains thereof,, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity.

The amino acid sequence of human Dkk-1 is available under GenBank reference Swiss protein accession number AAF02674.1 (GI: 6049604) and the encoding nucleic acid under reference AF177394.1 (GI: 6049603) . The amino acid sequence of human Dkk-2 is available under GenBank reference Swiss protein accession number AAQ88780.1 (GI :37181953) and the encoding nucleic acid under reference AY358414.1 (GI:37181952) .

The amino acid sequence of human Dkk-3 is available under GenBank reference Swiss protein accession number AAF02676.1 (GI:6049608) and the encoding nucleic acid under reference AF177396.1 (GI:6049607) .

The amino acid sequence of human Dkk-4 is available under GenBank reference Swiss protein accession number AAF02677.1 (GI: 6049610) and the encoding nucleic acid under reference AF177397.1 (GI: 6049609) .

Dkk receptors which bind Dkk polypeptides include LRP polypeptides, such as LRP-5 and LRP-6 polypeptides and Kremen polypeptides, such as Kremen 1 and Kremen 2 polypeptides .

An LRP polypeptide may comprise an LRP sequence from any mammalian species, for example a mouse LRP or a human LRP, or may comprise a sequence which is a fragment or variant thereof.

A Kremen polypeptide may comprise a Kremen sequence from any mammalian species, for example a mouse Kremen or a human Kremen, or may comprise a sequence which is a fragment or variant thereof.

A fragment or variant of a wild type LRP or Kremen sequence may differ from the wild-type sequence by the addition, deletion, substitution and/or insertion of one or more amino acids, provided the function of enhancing development of a dopaminergic neuronal fate in a stem cell, neural stem cell, embryonic stem cell or neural progenitor or precursor cell is retained. (Monaghan et al. (1999), Bafico et al. , (2001); Li et al . (2002); MacDonald et al. (2004); Mao et al. (2001a) ; Semenov et al . , (2001) .

For example, a LRP polypeptide may comprise an amino acid sequence which shares greater than about 30% sequence identity with the extracellular domain or the full length sequence of human LRP-5 or LRP-6, greater than about 40%, greater than about 45%, greater than about 55%, greater than about 65%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%. The sequence may share greater than about 30% similarity with the extracellular domain or the full length sequence of human LRP-5 or LRP-6, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity.

The amino acid sequence of human LRP-5 amino acid sequence is available under GenBank reference accession number NP_002326.1 (GI:4505019) and encoding nucleic acid under reference NM_002335.1 (GI:4505018) for DNA. The extracellular domain of homo sapiens LRP-5 is encoding by a 4100 base pair region starting at the initial ATG.

The amino acid sequence of human LRP-6 amino acid sequence is available under GenBank reference accession number NP_002327.1 (GI:4505017) and encoding nucleic acid under reference NM_002336.1

(GI:4505016) for DNA. The extracellular domain of homo sapiens LRP-6 is encoding by a 4167 base pair region starting at the initial ATG.

A Kremen polypeptide may comprise an amino acid sequence which shares greater than about 30% sequence identity with the extracellular domain or the full length sequence of human Kremen-1 or Kremen-2, greater than about 40%, greater than about 45%, greater than about 55%, greater than about 65%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%. The sequence may share greater than about 30% similarity with the extracellular domain or the full length sequence of human Kremen-1 or Kremen-2, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity.

The amino acid sequence of human Kremen-1 amino acid sequence is available under GenBank reference accession number NP_114434.3 (GI :24041012) and encoding nucleic acid under reference NM_032045.3 (GI:24041011) for DNA.

The amino acid sequence of human Kremen-2 amino acid sequence is available under GenBank reference accession number NP_078783.1 (GI :13375642) and encoding nucleic acid under reference NM_024507.2 (GI:27437002) for DNA.

In some preferred embodiments, an LRP or Kremen polypeptide may comprise or consist of the extracellular domain of human LRP or Kremen or an amino acid sequence which is a fragment or variant of the extracellular domain of human LRP or Kremen.

In some embodiments, the LRP or Kremen polypeptide may be a soluble, non-membrane bound polypeptide. The polypeptide may, for example, lack the transmembrane and/or intracellular domains of the wild-type LRP or Kremen.

In other embodiments, the LRP or Kremen polypeptide may be membrane- bound. The polypeptide may, for example, comprise the transmembrane and/or intracellular domains of the wild-type LRP or Kremen. A Dkk ligand/receptor polypeptide as described herein may further comprise one or more additional amino acids. For example, the LRP or Kremen polypeptide may be a fusion comprising an LRP or Kremen amino acid sequence, for example the extracellular domain of human LRp or Kremen or a fragment or variant thereof, and a peptidyl moiety that may confer one or more additional properties. For example, the fusion may comprise an immunoglobulin Fc domain.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps . Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. MoI. Biol. 215: 405- 410) , FASTA (which uses the method of Pearson and Lipman (1988) PiVAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J^". MoI Biol. 147: 195-197), or the TBLASTN program, of Altschul et al . (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl . Acids Res. (1997) 25 3389-3402) may be used. Sequence identity and similarity may also be determined using Genomequest™ software (Gene- IT, Worcester MA USA) .

Sequence comparisons are preferably made over the full-length of the relevant sequence described herein.

Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. In some embodiments, the level and/or activity of a Dkk ligand/receptor polypeptide in a cell may be increased by treating the cell with a Dkk ligand, or a Dkk receptor. For example, the cell may be treated with a Dkk ligand/receptor polypeptide.

A cell may be treated with a Dkk ligand or Dkk receptor in vivo, ex vivo, or in culture (in vitro) .

In the methods described herein, treating with a Dkk ligand or receptor may be by means of contacting a cell with one or both polypeptides. Treating with a Dkk ligand or receptor may be by- means of provision of purified and/or recombinant Dkk-2 or -3 polypeptide or other Dkk receptor agonist and/or LRP-5/6 receptor or polypeptide to a culture comprising the stem, progenitor or precursor cell, or to such a cell in vivo. Treating with a Dkk ligand or receptor may comprise introducing one or more copies of the encoding nucleic acid or protein into the cell by protein transduction. Methods of transforming cells with nucleic acid and introducing proteins into cells are described further below. Contacting with a Dkk ligand or receptor may be by means of providing in vivo or within a culture comprising the stem, progenitor or precursor cell or neuronal cell, a cell that produces the polypeptide (s) . The cell that produces the Dkk ligand or receptor may be a recombinant host cell that produces the polypeptide by recombinant expression. A co-cultured host cell may be transformed with nucleic acid encoding the Dkk ligand or receptor, and/or the co-cultured cell may contain introduced Dkk ligand or receptor. Dkk ligand or receptor protein, or encoding nucleic acid, may be introduced into the cell in accordance with available techniques in the art, examples of which are described below.

The co-cultured or host cell may be another stem, neural stem, progenitor, precursor or neural cell. Treatment with Dkk ligand or receptor may also be by means of up-regulating Dkk ligand or receptor expression in the cell or by down regulating or inhibiting an inhibitor molecule of Dkk ligand or receptor.

In other embodiments, the level and/or activity of a Dkk ligand/ receptor polypeptide such as Dkk-1 or Dkk-4 may be reduced by treating the cell with an inhibitor, antagonist, or suppressor of the polypeptide.

The level and/or activity of a Dkk ligand/receptor polypeptide such as Dkk-1 or Dkk-4, may also be reduced by repressing expression of the gene encoding the polypeptide or up-regulating or activating an inhibitor, blocker, antagonist or suppressor of the polypeptide.

Suitable inhibitors include antibodies and recombinant fragments which bind specifically to the Dkk polypeptide. Specific antibodies may be obtained using techniques that are standard in the art.

Suitable suppressors include anti-sense and sense nucleic acids (e.g. RNAi) . The use of suppressor nucleic acids is well known in the art (see for example Peyman and Ulman, Chemical Reviews, 90:543- 584, (1990); Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992); Reynolds et al Nature Biotech (2004) 22 3 326; Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001); Zamore PD et al Cell, 101, 25-33, (2000); Elbashir SM. et al . Nature, 411, 494-498, (2001) ) .

The level and/or activity of a Dkk ligand/ receptor polypeptide such as Dkk-1 or Dkk-4 may also be reduced by ^λknocking out' the relevant gene in an embryonic stem, neural stem, progenitor or precursor cell using standard recombinant techniques.

In other embodiments, the level and/or activity of a Dkk ligand/receptor polypeptide may be modulated by modulating the activity of a proprotein convertase in said cell. Examples of proprotein convertases include proteases which specifically cleave the sequence Arg-X-X-Arg, Arg/Lys-X-X-X-Arg/Lys- Arg, or Arg/Lys-Arg, such as furin or protease convertase 2 (PC2) .

The activity of a proprotein convertase may be reduced by treating the cell with an inhibitor or antagonist or increased by treating the cell with an agonist or co-factor.

A "stem cell" is any cell type that can self renew and, if it is an embryonic stem (ES) cell, can give rise to all cells in an individual, or, if it is a multipotent or neural stem cell, can give rise to all cell types in the nervous system, including neurons, astrocytes and oligodendrocytes. A stem cell may express one or more of the following markers: Oct-4; Nanog; Soxl-3; stage specific embryonic antigens (SSEA-I, -3, and -4), and the tumor rejection antigens TRA-1-60 and -1-81, as described (Tropepe et al . 2001; Xu et al . , 2001) . A neural stem cell may express one or more of the following markers: Nestin; the p75 neurotrophin receptor; Notchl, SSEA-I; Sox2; NCAM; RC2 (Capela and Temple, 2002) .

A "neural progenitor cell" is a daughter or descendant of a neural stem cell, with a more differentiated phenotype and/or a more reduced differentiation potential compared to the stem cell. A - precursor cell is any other cell being in a direct lineage relation with neurons during development or not but that under defined environmental conditions¹ can be induced to transdifferentiate or redifferentiate or acquire a neuronal phenotype. In preferred embodiments, the stem, neural stem, progenitor, precursor or neural cell does not express or express low levels of tyrosine hydroxylase either spontaneously or upon deprivation of mitogens (e.g. bFGF, EGF or serum) .

A stem cell, neural stem cell or neural progenitor or precursor cell may be obtained or derived from any embryonic, fetal or adult tissue, including bone marrow, skin, eye, nasal epithelia, or umbilical cord, or region of the nervous system, e.g. from the cerebellum, the ventricular zone, the sub-ventricular zone, the striatum, the midbrain, the hindbrain, the cerebral cortex or the hippocampus. It may be obtained or derived from a vertebrate organism, e.g. from a mammal, which may be human or non-human, such as rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle, horse, or primate, from a bird, such as a chicken, or from an amphibian.

In preferred embodiments of the present invention, adult stem/progenitor/precursor cells are used, in vitro, ex vivo or in vivo. This requires a consenting adult (e.g. from which the cells are obtained) and approval by the appropriate ethical committee. If a human embryo/fetus is used as a source, the human embryo is one that would otherwise be destroyed without use, or stored indefinitely, especially a human embryo created for the purpose of IVF treatment for a couple having difficulty conceiving. IVF generally involves creation of human e'mbryos in a number greater than the number used for implantation and ultimately pregnancy. Such spare embryos may commonly be destroyed. With appropriate consent from the people concerned, in particular the relevant egg donor and/or sperm donor, an embryo that would otherwise be destroyed can be used in an ethically positive way to the benefit of sufferers of severe neurodegenerative disorders such as Parkinson' s disease. The present invention itself does not concern the use of a human embryo in any stage of its development. As noted, the present invention minimizes the possible need to employ a material derived directly from a human embryo, whilst allowing for development of valuable therapies for terrible diseases. Any therapeutic interventions based on the present invention must also be performed according to the relevant national laws and ethical guidelines.

In some preferred embodiments, a stem or progenitor or precursor cell contacted with a Dkk ligand or a Dkk receptor and otherwise treated and/or used in accordance with any aspect of the present invention is obtained from a consenting adult or child for which appropriate consent is given, e.g. a patient with a disorder that is subsequently treated by transplantation back into the patient of neurons generated in accordance with the invention, and/or treated with a Dkk ligand or Dkk receptor, for example a Dkk polypeptide or LRP polypeptide as described above, to promote or induce endogenous dopaminergic neuron development or function.

The stem or progenitor or precursor cell in which a neuronal fate is induced may exhibit an undifferentiated phenotype or a primitive neuronal phenotype. It may be a totipotent cell, capable of giving rise to any cell type in an individual, or a multipotent cell which is capable of giving rise to a plurality of distinct neuronal phenotypes, or a precursor or progenitor cell, capable of giving rise to more limited phenotype during normal development but capable of giving rise to other cells when exposed to appropriate environmental factors in vitro. It may lack markers associated with specific neuronal fates, e.g. tyrosine hydroxylase.

A cell may be Nurrl positive (i.e. it expresses Nurrl) or Nurrl negative (i.e. it does not express Nurrl) . In some embodiments, a cell which is Nurrl negative may spontaneously express Nurrl during the differentiation process.

In some embodiments, the stem or progenitor or precursor cell in which a neuronal fate is induced may be under oxidative stress. Neurons produced from such cells may be useful as disease cell models. Oxidative stress may be imposed on a stem or progenitor or precursor cell using conventional toxin-based or genetic means.

In a method of inducing a neuronal fate wherein the cells are treated with a Dkk ligand or a Dkk receptor, a majority of the cells may be induced to adopt a neuronal fate. Dopaminergic induction or differentiation may be enhanced in neuronal cells. In preferred embodiments, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% of the stem and/or progenitor cells may be induced to a neuronal fate.

A Dkk ligand/receptor polypeptide may be expressed from encoding nucleic acid either in situ in a stem, or neural stem, precursor or progenitor cell or neuronal cell or in vitro in an expression system prior to isolation and purification. Transformed Dkk and/or LRP nucleic acid may be contained on an extra-genomic vector or it may be incorporated, preferably stably, into the genome. It may be operably-linked to a promoter which drives its expression above basal levels in stem cells, or neural stem, precursor or progenitor cells, or neuronal cells, as is discussed in more detail below.

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.

Methods of introducing genes into cells are well known to those skilled in the art. Vectors may be used to introduce Dkk and/or LRP encoding nucleic acid into stem, or neural stem, precursor or progenitor cells or neuronal cells, whether or not the nucleic acid remains on the vector or is incorporated into the genome. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences. Vectors may contain marker genes and other sequences as appropriate. The regulatory sequences may drive expression of Dkk and/or LRP encoding nucleic acid within the stem, or neural stem, precursor or progenitor cells or neural cells. For example, the vector may be an extra-genomic expression vector, or the regulatory sequences may be incorporated into the genome with Dkk and/or LRP encoding nucleic acid. Vectors may be plasmids or viral.

Dkk ligand/receptor polypeptide encoding nucleic acid may be placed under the control of an externally inducible gene promoter to place it under the control of the user. The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus. The nature of the stimulus varies between promoters . Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. An example of an inducible promoter is the Tetracyclin ON/OFF system (Gossen, et al . , 1995) in which gene expression is regulated by tetracyclin analogs.

For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold

Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology,

Ausubel et al. eds . , John Wiley & Sons, 1992 or later edition.

Marker genes such as antibiotic resistance or sensitivity genes or fluorescent reporters may be used in identifying clones containing nucleic acid of interest, as is well known in the art. Clones may also be identified or further investigated by binding studies, Southern blot hybridisation, immunohistochemistry, PCR and other techniques to detect protein or nucleic acid expression in the cells .

Nucleic acid encoding a Dkk receptor/ligand polypeptide such as a Dkk or LRP polypeptide may be integrated into the genome of the host stem, neural stem, progenitor, precursor or neural cell. Integration may be promoted by including in the transformed nucleic acid sequences which promote recombination with the genome, in accordance with standard techniques. The integrated nucleic acid may include regulatory sequences able to drive expression of the Dkk and/or LRP encoding nucleic acid in a stem cell, or neural stem, progenitor or precursor cells, or neuronal cells. The nucleic acid may include sequences which direct its integration to a site in the genome where the Dkk and/or LRP coding sequence will fall under the control of regulatory elements able to drive and/or control its expression within the stem, or neural stem, precursor or progenitor cell, or neuronal cell. The integrated nucleic acid may be derived from a vector used to transform Dkk and/or LRP nucleic acid into the stem cell, or neural stem, precursor or progenitor cells, or neuronal cells, as discussed herein.

The introduction of nucleic acid comprising encoding Dkk and/or LRP sequence, whether that nucleic acid is linear, branched or circular, may be generally referred to without limitation as "transformation". It may employ any available technique. Suitable techniques may include calcium phosphate transfection, DEAE-Dextran, PEI, electroporation, mechanical techniques such as microinjection, direct DNA uptake, receptor mediated DNA transfer, transduction using retrovirus or other virus and liposome-, lipid- or other cationic carrier- mediated transfection. When introducing a chosen gene construct into a cell, certain considerations must be taken into account, well known to those skilled in the art. It will be apparent to the skilled person that the particular choice of method of transformation to introduce a Dkk receptor/ligand polypeptide, such as Dkk and/or LRP polypeptide into a stem cell, or neural stem, precursor or progenitor cells or a neuronal cell is not essential to or a limitation of the invention.

Suitable vectors and techniques for in vivo transformation of stem cells, or neural stem, precursor or progenitor cells or neuronal cells with Dkk and/or LRP nucleic acid are well known to those skilled in the art. Suitable vectors include adenovirus, adeno- associated virus papovavirus, vaccinia virus, herpes virus, lentiviruses and retroviruses. Disabled virus vectors may be produced in helper cell lines in which genes required for production of infectious viral particles are expressed. Suitable helper cell lines are well known to those skilled in the art. By way of example, see: Fallaux, F.J., et al . , (1996) Hum Gene Ther 7(2), 215-222; Willenbrink, W., et al. , (1994) J Virol 68(12), 8413-8417; Cosset, F.L., et al., (1993) Virology 193(1), 385-395; Highkin, M.K., et al., (1991) Poult Sci 70(4), 970-981; Dougherty, J.P., et al . , (1989) J Virol 63(7) , 3209-3212; Salmons, B., et al. , (1989) Biochem Biophys Res Commun 159(3) , 1191-1198; Sorge, J., et al . , (1984) MoI Cell Biol 4(9), 1730-1737; Wang, S., et al. , (1997) Gene Ther 4(11), 1132-1141; Moore, K.W., et al . , (1990) Science 248(4960), 1230-1234; Reiss, CS. , et al . , (1987) J Immunol 139(3), 711-714. Helper cell lines are generally missing a sequence which is recognised by the mechanism which packages the viral genome. They produce virions which contain no nucleic acid. A viral vector which contains an intact packaging signal along with the gene or other sequence to be delivered (e.g. Dkk and/or LRP coding sequence) is packaged in the helper cells into infectious virion particles, which may then be used for gene delivery to stem cells, or neural stem, precursor or progenitor cells or neuronal cells.

As an alternative or addition to increasing transcription and/or translation of endogenous Dkk and/or LRP, expression of Dkk and/or LRP above basal levels may be caused by introduction of one or more extra copies of Dkk and/or LRP protein into the stem, neural stem, precursor, progenitor or neural cell by microinjection or other carrier-based or protein delivery or transduction system, including cell penetrating peptides, i.e. TAT, transportan, Antennapedia penetratin peptides (Lindsay 2002) .

The present invention allows for generation of large numbers of dopaminergic neurons . These dopaminergic neurons may be used as source material to replace cells which degenerate or are damaged or lost in Parkinson's disease. Preferably, the cell is mitotic when it is contacted with the Dkk ligand or Dkk receptor.

In methods of the invention, the cell may additionally be contacted with one or more agents selected from: basic fibroblast growth factor (bFGF) ; epidermal growth factor (EGF) ; and an activator of the retinoid X receptor (RXR) , e.g. the synthetic retinoid analog SR11237, (Gendimenico, G. J., et al . , (1994) J Invest Dermatol 102(5), 676-80), 9-cis retinol or docosahexanoic acid (DHA) or LG849 (Mata de Urquiza et al . , 2000); a member of the Wnt family of ligands, including Wnt-1, -3a and -5a (Catello-Branco et al . , 2003); an upstream regulator of Wnts, such as Msx-1 (Her and Abate-Shen, Biochemical And Biophysical Research Communications 227, 257-265 (1996) ; Shang et al . , Proc. Natl. Acad. Sci. USA 91, 118-122 (1994); or b-catenin (Catello-Branco et al . , 2003); or Nurrl (Wagner et al. , 1999) . Treating cells in accordance with the invention with one or more of these agents may be used to increase the proportion of the stem, progenitor or precursor cells, which adopt a dopaminergic fate, or enhance dopaminergic induction or differentiation in a neuronal cell. The method of inducing a dopaminergic fate or enhancing dopaminergic induction or differentiation in a neuronal cell in accordance with the present invention may include contacting the cell with a member of the FGF family of growth factors, e.g. FGF4, FGF8 or FGF20, for example in a pre-treatment step.

Advantageously, the cells may be contacted with two or more of the above agents .

Instead or as well as pretreating, the additional factors may be to treat cells simultaneously with Dkk and/or LRP treatment.

A method according to the invention in which a neuronal fate is induced in a stem, neural stem or progenitor or precursor cell or there is enhanced dopaminergic induction or differentiation in a neuronal cell, may include detecting a marker for the neuronal fate, beta-tubulin III (TuJl) is one marker of the neuronal fate (Menezes, J. R., et al., (1994) J Neurosci 14(9), 5399-5416) . Other neuronal markers include neurofilament and MAP2. If a particular neuronal phenotype is induced, the marker should be specific for that phenotype. For the dopaminergic fate, expression of tyrosine hydroxylase (TH) , aromatic L-amino acid decarboxylase (AADC) , dopamine transporter (DAT) and dopamine receptors may be detected e.g. by immunoreactivity or in situ hybridization. Tyrosine hydroxylase is a major marker for DA cells. Contents and/or release of dopamine and metabolites may be detected e.g. by High Pressure Liquid Chromatography (HPLC) (Cooper, J. R., et al . , The Biochemical Basis of Neuropharmacology, 7th Edition, (1996) Oxford University Press) . The absence of Dopamine β hydroxylase and GABA or GAD (in the presence of TH/dopamine/DAT) is also indicative of dopaminergic fate. Additional markers include Nurrl, Retinaldehyde dehydrogenasel (Raldhl) or Aldehyde dehydrogenase type 2 (AHD-2) , GIRK2, Lmxla/b and Pitx3.

Detection of a marker may be carried out according to any method known to those skilled in the art. The detection method may employ a specific binding member capable of binding to a nucleic acid sequence encoding the marker, the specific binding member comprising a nucleic acid probe hybridisable with the sequence, or an immunoglobulin/antibody domain with specificity for the nucleic acid sequence or the polypeptide encoded by it, the specific binding member being labelled so that binding of the specific binding member to the sequence or polypeptide is detectable. A "specific binding member" has a particular specificity for the marker and in normal conditions binds to the marker in preference to other species. Alternatively, where the marker is a specific mRNA, it may be detected by binding to specific oligonucleotide primers and amplification in e.g. the polymerase chain reaction. Nucleic acid probes and primers may hybridize with the marker under stringent conditions. Suitable conditions include, e.g. for detection of marker sequences that are about 80-90% identical, hybridization overnight at 42C in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55C in 0.IX SSC, 0.1% SDS. For detection of marker sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65C in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 6OC in 0. IX SSC, 0.1% SDS.

In a further aspect, the present invention provides a neuron produced in accordance with any one of the methods disclosed herein. The neuron may have a primitive neuronal phenotype. It may be capable of giving rise to a plurality of distinct neuronal phenotypes . The neuron may have a particular neuronal phenotype. In preferred embodiments, the neuron has a dopaminergic phenotype.

The neuron may contain nucleic acid encoding a molecule with neuroprotective or neuroregenerative properties operably linked to a promoter which is capable of driving expression of the molecule in the neuron. The promoter may be an inducible promoter, e.g. the TetON chimeric promoter, so that any damaging over-expression may be prevented. The promoter may be associated with a specific neuronal phenotype, e.g. the TH promoter, the Nurrl promoter, the dopamine transporter promoter or the Pitx3 promoter.

The encoded molecule may be such that its expression renders the neuron independent of its environment, i.e. such that its survival is not dependent on the presence of one or more factors or conditions in e.g. the neural environment into which it is to be implanted. By way of example, the neuron may contain nucleic acid encoding one or more of the neuroprotective or neuroregenerative molecules described below operably linked to a promoter that is capable of driving expression of the molecule in the neuron. In addition or alternatively, expression of the encoded molecule may function in neuroprotection or neuroregeneration of the cellular environment surrounding that neuron. In this way, the neuron may be used in a combined cell and gene therapy approach to deliver molecules with neuroprotective and neuroregenerative properties.

Examples of molecules with neuroprotective and neuroregenerative properties include:

(i) neurotropic factors able to compensate for and prevent neurodegeneration. One example is glial derived neurotropic growth factor (GDNF) which is a potent neural survival factor, promotes sprouting from dopaminergic neurons and increases tyrosine hydroxylase expression (Tomac, et al., (1995) Nature, 373, 335-339; Arenas, et al . , (1995) Neuron, 15,1465-1473) . By enhancing axonal elongation GDNF, GDNF may increase the ability of the neurons to inervate their local environment. Other neurotropic molecules of the GDNF family include Neurturin, Persephin and Artemin. Neurotropic molecules of the neurotropin family include nerve growth factor (NGF) , brain derived neurotropic factor (BDNF) , and neurotropin-3, -4/5 and -6. Other factors with neurotrophic activity include members of the FGF family for instance FGF2, 4, 8 and 20; members of the Wnt family, including Wnt-1, -2, -5a, -3a and 7a,- members of the BMP family, including BMP2, 4, 5 and 7, nodal, activins and GDF; and members of the TGFalpha/beta family.

(ii) anti-apoptotic molecules. Bcl2 which plays a central role in cell death. Over-expression of Bcl2 protects neurons from naturally occurring cell death and ischemia (Martinou, et al . , (1994) Neuron, 1017-1030) . Another anti-apoptotic molecule specific for neurons is BcIX-L.

(iii) axon regenerating and/or elongating and/or guiding molecules which assist the neuron in innervating and forming connections with its environment, e.g. ephrins. Ephrins define a class of membrane- bound ligands capable of activating tyrosine kinase receptors . Ephrins have been implicated in neural development (Irving, et al . , (1996) Dev. Biol., 173, 26-38; Krull, et al . , (1997) Curr. Biol. 7, 571-580; Frisen, et al. , (1998) Neuron, 20, 235-243; Gao, et al. , (1996) PNAS, 93, 11161-11166; Torres, et al. , (1998) Neuron, 21,

1453-1463; Winslow, et al . , (1995) Neuron, 14, 973-981; Yue, et al . , (1999) J Neurosci 19(6), 2090-2101.

(iv) transcription factors, e.g. the homeobox domain protein Ptx3 (Smidt, M. P., et al. , (1997) Proc Natl Acad Sci USA, 94(24), 13305- 13310) , Lmxla/b, Pax2, Pax5, Pax8, or engrailed 1 or 2 (Wurst and Bally-Cuif, 2001; Rhinn and Brand, 2001) ; or upstream regulators of Wnts, including as Msx-1 (Her and Abate-Shen, 1996; Shang et al. , 1994); or beta-catenin (Catello-Branco et al. , 2003); or Nurrl (Wagner et al. , 1999); or neurogenic genes of the basic helix-loop- helix family.

A neuron in accordance with or for use in the present invention may^¬ be substantially free from one or more other cell types, e.g. from stem, neural stem, precursor or progenitor cells. Neurons may be separated from neural stem or progenitor cells using any technique known to those skilled in the art, including those based on the recognition of extracellular epitopes by antibodies and magnetic beads or fluorescence activated cell sorting (FACS) . By way of example, antibodies against extracellular regions of molecules found on stem, neural stem, precursor or progenitor cells but not on neurons may be employed. Such molecules include Notch 1, CD133, SSEAl, promininl/2, RPTPβ/phosphocan, TIS21 and the glial cell line derived neurotrophic factor receptors GFR alphas or NCAM. Stem cells bound to antibodies may be lysed by exposure to complement, or separated by, e.g. magnetic sorting (Johansson, et al . , (1999) Cell, 96, 25-34) . If antibodies which are xenogeneic to the intended recipient of the neurons are used, then any e.g. stem, neural stem or progenitor or precursor cells which escape such a cell sorting procedure are labelled with xenogeneic antibodies and are prime targets for the recipient's immune system. Alternatively, cells that acquire the desired phenotype could also be separated by antibodies against extracellular epitopes or by the expression of transgenes including fluorescent proteins under the control of a cell type specific promoter. By way of example dopaminergic neurons could be isolated with fluorescent proteins expressed under the control of TH, DAT, Ptx3 or other promoters specifically used by dopaminergic neurons.

Methods of the invention may comprise additional negative or positive selection methods to enrich for neural stem, progenitor or precursor cells, or other stem or neural cells with the desired phenotype.

Negative selection may be used to enrich for DA neurons. Selective neurotoxins for non-DA neurons may be used, for instance 5-7- dihydroxytryptamine (to eliminate serotoninergic neurons) , or antibodies coupled to saponin or a toxin or after addition of complement, for instance antibodies against GABA transporter (to eliminate GABAergic neurons) . Methods of the invention may comprise additionally treating or contacting a neural stem, progenitor or precursor cell, or other stem or neural cell with a negative selection agent, preferably in vitro, e.g. by adding the negative selection agent to an in vitro culture containing the cell, or by culturing the cell in the presence of the negative selection agent. A negative selection agent selects against cell types other than the desired cell type(s) . For example, where the invention relates to promoting, enhancing or inducing a dopaminergic neuronal phenotype, the negative selection agent may select against cells other than DA neurons and cells that develop into DA neurons such as stem cells and neural stem, precursor and progenitor cells. Thus, the negative selection agent may select against differentiated cells with a non- DA phenotype, such as non-DA neurons. The negative selection agent may reduce or prevent proliferation of and/or kill cells other than the desired cell type(s) . The negative selection agent may be a selective neurotoxin that reduces the population of neurons other than DA neurons. For example, the negative selection agent may be 5-7-dihydroxytryptamine (to reduce serotoninergic neurons) . The negative selection agent may be an antibody or antibody fragment specific for a non-DA neuron, wherein the antibody or antibody fragment (e.g. scFv or Fab) is a blocking antibody or is coupled to saponin or to a toxin. For instance the antibody may be a blocking antibody against FGF-4 (to reduce serotonin neurons) , or an antibody specific for GABA transporter coupled to a toxin (to reduce GABAergic neurons) .

In methods of the invention, the neural stem, progenitor or precursor cell or other stem or neural cell may be grown in the presence of an antioxidant (e.g. ascorbic acid), low oxygen tension and/or a hypoxia-induced factor (e.g. HIF or erythropoetin) .

The present invention further provides in various aspects and embodiments the use of an agent selected from a Dkk ligand and/or Dkk receptor, such as a Dkk polypeptide and/or a LRP polypeptide, or nucleic acid encoding a Dkk ligand or a Dkk receptor, or a synthetic Dkk ligand and/or Dkk receptor analogue, or a protein, nucleic acid or synthetic drug working to enhance, switch or modulate one or more signalling components downstream of Dkk and/or LRP, in therapeutic methods comprising administering the Dkk ligand and/or Dkk receptor, such as a Dkk and/or LRP polypeptide, or encoding nucleic acid or other said agent to an individual to induce, promote or enhance dopaminergic neuron development in the brain by acting on either endogenous or on exogenously supplied stem, progenitor or precursor cells, or neuronal cells, and/or to inhibit or prevent loss or promote the survival or phenotypic differentiation or maturation, or neuritogenesis, or synaptogenesis, or neurotransmission, or functional output of dopaminergic neurons, e.g. in treatment of an individual with a Parkinsonian syndrome or Parkinson's disease. A Dkk ligand and/or Dkk receptor, such as a Dkk and/or LRP polypeptide, or encoding nucleic acid or other said agent may be administered in any suitable composition, e.g. comprising a pharmaceutically acceptable excipient or carrier, and may be used in the manufacture of a medicament for treatment of a neurodenerative disorder, Parkinsonian syndrome or Parkinson's disease. A Dkk ligand and/or Dkk receptor, such as a Dkk and/or LRP polypeptide, or encoding nucleic acid may be administered to or targeted to the central nervous system and/or brain.

The present invention extends in various aspects not only to a neuron produced in accordance with any one of the methods disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a neuron, stem, progenitor or precursor cell, use of such a neuron, stem, progenitor or precursor cell or neuronal cell or composition in a method of medical treatment, a method comprising administration of such a neuron, stem, progenitor, precursor or neuronal cell or composition to a patient, e.g. for treatment (which may include preventative treatment) of Parkinson's disease or other (e.g. neurodegenerative) diseases, use of such a neuron or cell in the manufacture of a composition for administration, e.g. for treatment of Parkinson's disease or other (e.g. neurodegenerative diseases), and a method of making a pharmaceutical composition comprising admixing such a neuron or cell with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients, e.g. a neuroprotective molecule, a neuroregenerative molecule, a retinoid, growth factor, astrocyte/glial cell, anti-apoptotic factor, or factor that regulates gene expression in stem, progenitor or precursor cells or neuronal cells or in the host brain. Such optional ingredients may render the neuron independent of its environment, i.e. such that its survival is not dependent on the presence of one or more factors or conditions in its environment. By way of example, the method of making a pharmaceutical composition may include admixing the neuron with one or more factors found in the developing ventral mesencephalon. The neuron may be admixed with GDNF and/or neurturin (NTN) . The present invention provides a composition containing a neuron, stem, progenitor or precursor cell or neuronal cell produced in accordance with the invention, and one or more additional components. Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the neuron or cell, a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabiliser, anti-oxidant or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the activity of the neuron. The precise nature of the carrier or other material will depend on the route of administration. The composition may include one or more of a neuroprotective molecule, a neuroregenerative molecule, a retinoid, growth factor, astrocyte/glial cell, or factor that regulates gene expression in stem, neural stem, precursor or progenitor cells or neuronal cells. Such substances may render the neuron independent of its environment as discussed above.

Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

The composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to.prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection. A composition may be prepared using artificial cerebrospinal fluid. The present invention extends to the use of a neuron produced in accordance with the invention in a method of medical treatment, particularly the treatment of a medical condition associated with degeneration, damage to, the loss of, or a disorder in neuronal cells. Moreover, the invention may provide the use of a neuron of a specific phenotype in the treatment of a condition, disease or disorder, which is associated with generation, damage to, or the loss of neurons of that phenotype. More particularly, the invention provides the use of a dopaminergic neuron in the treatment of human Parkinson's disease. While the invention particularly relates to materials and methods for treatment of neurodegenerative diseases

(e.g. α-synucleinopathies such as Parkinson's disease), it is not limited thereto. By way of example, the invention extends to the treatment of any pathology or disease affecting dopaminergic neurons and degeneration in or damage to other regions of the nervous system, in particular regions containing Nurrl⁺ cells, such as the spinal cord and/or cerebral cortex.

In methods of treatment in which the administered cell is a stem, progenitor or precursor that is capable of giving rise to two or more distinct neuronal phenotypes, the neuron, cell or composition may be introduced into a region containing astrocytes/glial cells which direct the differentiation of the cell to a desired specific neuronal fate. The cell or composition may, for example, be injected into the ventral mesencephalon where it may interact with Type 1 astrocytes/glial cells and be induced to adopt a dopaminergic phenotype. Alternatively or in addition, an implanted composition may contain a neuron or cell in combination with one or more factors which direct its development toward a specific neuronal fate as discussed above, e.g. with a Type 1 astrocyte/glial cell.

Cells may be implanted into a patient by any technique known in the art (e.g. Lindvall, 0., (1998) Mov. Disord. 13, Suppl . 1:83-7; Freed, CR. , et al . , (1997) Cell Transplant, 6, 201-202; Kordower, et al., (1995) New England Journal of Medicine, 332, 1118-1124; Freed, C.R., (1992) New England Journal of Medicine, 327, 1549-1555) .

Administration of a composition in accordance with the present invention is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

The methods provided herein may be carried out using primary cells in vivo or in vitro or cell lines as a source material. The advantage of cells expanded in vitro is that there is virtually no limitation on the number of neurons which may be produced.

In order to ameliorate possible disadvantages associated with immunological rejection of transplanted cells, stem or progenitor or precursor cells may be isolated from a patient and induced to the desired phenotype. Cells may then be transplanted to the patient. Advantageously, isolated stem or progenitor or precursor cells may be used to establish cell lines so that large numbers of immunocompatible neuronal cells may be produced. A further option is to establish a bank of cells covering a range of immunological compatibilities from which an appropriate choice can be made for an individual patient . Stem, neural stem, precursor or progenitor cells or neuronal cells derived from one individual may be altered to ameliorate or prevent rejection when they or their progeny are introduced into a second individual. By way of example, one or more MHC alleles in a donor cell may be replaced with those of a recipient, e.g. by homologous recombination.

If cells derived from a cell line carrying an immortalizing oncogene or reporter gene are used for implantation into a patient, the oncogene may be removed using the CRE-loxP system prior to implantation of the cells into a patient (Westerman, K. A. et al Proc. Natl. Acad Sci . USA 93, 8971 (1996)) . An immortalizing oncogene which is inactive at the body temperature of the patient may be used.

In a further aspect the present invention extends to the use of a cell or neuron produced in accordance with the invention in a method of screening for an agent for use in the treatment of a neurodegenerative disease. The neuron may be a dopaminergic neuron. The neurodegenerative disease may be an α-synucleinopathy such as Parkinsonian syndrome or Parkinson's disease. The agent may be a neuroprotective and/or neuroregenerative molecule and/or a developmental soluble signal and/or a factor or factors derived from ventral mesencephalic type one astrocytes or glial cells. The method may be carried out in vitro or in vivo.

The method may include:

(i) treating a neuron of the invention with a toxin for said neuron,- (ii) separating the neuron from the toxin;

(iii) bringing the treated neuron into contact with a test agent or test agents;

(iv) determining the ability of the neuron to recover from the toxin; (v) comparing said ability of the neuron to recover from the toxin with the ability of the or an identical neuron to recover from the toxin in the absence of contact with the test agent (s) .

The method may include: (i) treating a neuron of the invention with a toxin for the neuron in the presence of a test agent or test agents,-

(ii) determining the ability of the neuron to tolerate the toxin; (iii) comparing said ability of the neuron to tolerate the toxin with the ability of the or an identical neuron to tolerate the toxin in the absence of contact with the test agent (s) .

The toxin may be 6-hydroxydopamine, 5, 7-dihydroxytryptamine or 1- methyl-4-phenyl-1, 2, 3 , 6-tetrahydropyridine (MPTP) , proteasome inhibitors, including lactacystin, or pesticides, including rotenone, all of which lead to the death of catecholaminergic neurons and experimentally reproduce features of Parkinson' s disease. The ability of the neuron to recover from or tolerate the toxin may be determined by any method known to those skilled in the art, for example by monitoring cell viability or cell loss, (e.g. by cell counting, e.g. by the TUNEL technique) or active caspase-3 immunostaining, by monitoring morphology, (e.g. sprouting, axonal elongation and/or branching) , and/or by monitoring biochemistry, (e.g. TH activity, e.g. neurotransmitter uptake/release/content) .

A method of screening for an agent for use in the treatment of a neurodegenerative disease may include;

(i) bringing a dopaminergic neuron produced by a method described above into contact with a test agent or test agents; (ii) determining the growth, function, maintenance, viability or neurotransmitter release of the dopaminergic neuron in the presence of said agent or agents .

In other embodiments, methods of screening for an agent for use in the treatment of a neurodegenerative disease may employ a neuron generated from a stem, progenitor or precursor cell which is under oxidative stress.

(i) providing a neuron which is generated from a stem, progenitor or precursor cell under oxidative stress, (ii) bringing the neuron into contact with a test agent or test agents,-

(iii) determining the growth, function, maintenance, viability or neurotransmitter release of the neuron in the presence of said agent or agents.

The growth, function, maintenance, viability or neurotransmitter release of the neuron in the presence of said agent or agents may be compared with the growth, function, maintenance, viability or neurotransmitter release of the neuron in the absence of said agent or agents. An increase in growth, neurotransmission, maintenance, or viability in the presence relative to the absence of agent or agents may be indicative that the agent or agents are useful in the treatment of a neurodegenerative disorder.

The growth, neurotransmission, or maintenance, or viability of the neuron may be determined by any method known to those skilled in the art, for example by cell counting, e.g. by the TUNEL technique or active caspase-3 immunostaining, by BrdU or ³H-Thymidine incorporation, by monitoring morphology, (e.g. sprouting, axonal elongation and/or branching) , and/or by monitoring biochemistry, (e.g. TH activity, e.g. neurotransmitter uptake/release/content), by gene or protein expression (e.g. immunohistochemistry, PCR, genechip, proteome) .

Examples of stem, neural stem, embryonic stem cell, precursor, progenitor or neural cells which may be used in the present invention include C17.2 (Snyder, E. Y. et al . Cell 68, 33-51 (1992)) and the H6 human cell line (Flax et al. Nature Biotech 16 (1998)) . Further examples of human neural and embryonic stem cells are described in: Gage, F.H., et al. Ann. Rev. Neurosci. 18 159-192 (1995); and Gotlieb (2002) Annu. Rev. Neurosci 25 381-407); Carpenter et al . Stem Cells. 5(1) : 79-88 (2003) ; and in http://stemcells .nih.gov/research/registry/ . While the present discussion has been made with reference to neural stem cells or neural progenitor or precursor cells, the methods provided herein may be applied to the induction of neuronal fates in other stem, progenitor or precursor cells. Examples of such cells include human and non-human embryonic stem cells—and stem cells associated with non-neural systems. The methods may be applied to stromal or hematopoietic stem cells and/or proliferative cells from the epidermis . Hematopoietic cells may be collected from blood or bone marrow biopsy. Stromal cells may be collected from bone marrow biopsy. Epithelial cells may be collected by skin biopsy or by scraping e.g. the oral mucosa. Since a neuronal phenotype is not a physiological in vivo fate of these stem, progenitor or precursor cells, the inductive process may be referred to as trans- differentiation, or de-differentiation and neural re- differentiation. A method of inducing such cells to a neuronal fate may include the use of microRNAs or anti-sense regulators to genes associated with non-neuronal phenotypes, i.e. to suppress and/or reverse the differentiation of these cells toward non-neuronal fates .

The methods of the present invention may be applied to stem cells not committed to a neural fate. They may be applied to stem cells which are capable of giving rise to two or more daughter stem cells associated with different developmental systems. Examples of these stem cells are embryonic stem cells, haematopoietic stem cells, proliferative cells from the epidermis, and neural stem cells.

In various further aspects the present invention is concerned with provision of assays and methods of screening for a factor or factors which enhance induction of a dopaminergic fate in a neural stem or progenitor or precursor cell or enhance dopaminergic induction or differentiation in a neuronal cell treated with a Dkk ligand such as a Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide. The invention provides a method of screening for a factor or factors able, either alone or in combination, to enhance, increase or potentiate induction of a dopaminergic fate in a stem, neural stem or progenitor or precursor cell or neuronal cell in the presence of Dkk and/or LRP. A further aspect of the present invention provides the use of a stem, embryonic stem, neural stem or progenitor or precursor cell or neuronal cell in the presence of Dkk and/or LRP in screening or searching for and/or obtaining/identifying a factor or factors which enhance induction of a dopaminergic fate in such a stem or progenitor or precursor cell or neuronal cell.

A method of screening may include:

(a) bringing a test substance into contact with a stem, neural stem or progenitor or precursor cell or neuronal cell in the presence of a Dkk ligand, such as a Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide, which contact may result in interaction between the test substance and the cell; and

(b) determining interaction between the test substance and the cell.

Interaction may be determined by determining the ability of the cell to bind, internalise the Dkk ligand/receptor polypeptide and/or activate signalling pathways or cellular processes leading to or accompanying increased dopaminergic cell numbers and/or differentiation. These include signalling processes regulated by kremen or LRP 5/6 receptors i.e. the regulation of beta-catenin or axin levels and/or degradation and/or translocation and/or phosphorylation as discussed herein.

A method of screening may include bringing a test substance into contact with a membrane fraction, soluble fraction or nuclear fraction derived from a stem, neural stem or progenitor or precursor cell or neuronal cell in the presence of Dkk ligand such as a Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide, and determining interaction between the test substance and the fraction. The preparation of these fractions is well within the capabilities of those skilled in the art.

Binding or interaction may be determined by any number of techniques known in the art, qualitative or quantitative. Interaction between the test substance and the stem or progenitor or neuronal cell may be studied by labelling either one with a detectable label and bringing it into contact with the other which may have been immobilised on a solid support, e.g. by using an antibody bound to a solid support, or via other technologies which are known per se including the Biacore system.

A screening method may include culturing a stem, neural stem or progenitor or precursor cell or neuronal cell in the presence of a test substance or test substances and analyzing the cell for differentiation to a dopaminergic phenotype, e.g. by detecting a marker of the dopaminergic phenotype as discussed herein. Tyrosine hydroxylase (TH) is one marker of the dopaminergic phenotype. Additional markers include Nurrl, Retinaldehyde dehydrogenasel

(Raldhl) or Aldehyde dehydrogenase type 2 (AHD-2) , GIRK2, Lmxla/b and Pitx3. The absence of Dopamine β hydroxylase and GABA or GAD (in the presence of TH/dopamine/DAT) is also indicative of dopaminergic fate.

Substances screened in accordance with the present invention may be natural or synthetic chemical compounds, including, for example, polypeptides, nucleic acids and small organic molecules.

A screening method may include comparing Type 1 astrocytes or early glial cells of the ventral mesencephalon with neural cells (e.g. astrocytes) which are unable to induce a dopaminergic fate in stem, neural stem or progenitor or precursor cells, in the presence of Dkk and/or LRP polypeptide. The comparison may for example be between Type 1 astrocytes or early glial cells during development of the ventral mesencephalon and Type 1 astrocytes or early glia from other neural locations .

A screening method involving astrocytes or early glial cells may employ immortalized astrocytes or immortalized glial cells. It may involve astrocyte cell lines or glial cell lines, e.g. astrocyte or glial mesencephalic cell lines. Such cell lines provide a homogenous cell population.

A screening method may employ any known method for analyzing a phenotypic difference between cells and may be at the DNA, mRNA, cDNA or polypeptide level. Differential screening and gene screening are two such techniques . A substance identified by any of the methods of screening described herein may be used as a test substance in any of the other screening methods described herein.

A screening method may employ a nucleic expression array, e.g. a mouse cDNA expression array. In this approach, an array of different nucleic acid molecules is arranged on a filter, quartz or another surface, e.g. by cross-linking the nucleic acid to the filter. A test solution or extract is obtained and the nucleic acid within it is labelled, e.g. by fluorescence. The solution or extract is then applied to the filter or genechip. Hybridisation of the test nucleic acid to nucleic acid on the filter or genechip is determined and compared to the hybridisation achieved with a control solution. A difference between the hybridisation obtained with the test and control samples is indicative of different nucleic acid content. For further information on nucleic acid arrays, see Clontech website (e.g. www.clontech.com) or Affymetrix website (e.g. www.affymetrix.com), findable using any available web browser.

A screening method may include comparing stem or progenitor or precursor or neural cells with stem or progenitor or precursor cells or neural cells in the presence of a Dkk ligand such as a Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide, e.g. to identify a factor or factors which enhance the proliferation and/or self-renewal and/or the differentiation and/or survival and/or promote the acquisition or the induction of a dopaminergic fate and/or induce dopaminergic neuron development and/or neurotransmission in stem, neural stem, precursor, progenitor or neural cells and/or enhance dopaminergic induction or differentiation in a neuronal cell in the presence of the Dkk ligand or Dkk receptor. Once the target gene(s) and/or factor(s) have been identified they may be isolated and/or purified and/or cloned and used in further methods.

A screening method may include culturing a stem, neural stem or progenitor or precursor cell or neuronal cell in the presence of a test substance or test substances and analyzing the cell for β- catenin activation, e.g. by dephosphorylation or reporter assays as discussed herein.

A screening method may include comparing stem or progenitor or precursor or neural cells with stem or progenitor or precursor cells or neural cells in the presence of a Dkk ligand such as a Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide, e.g. to identify a factor or factors which promote, increase or enhance β-catenin activation in stem, neural stem, precursor, progenitor or neural cells in the presence of the Dkk ligand or Dkk receptor. Once the target gene(s) and/or factor (s) have been identified they may be isolated and/or purified and/or cloned and used in further methods .

In a related aspect, the present invention provides a method of screening for a substance which modulates the ability of a Dkk ligand or Dkk receptor, such as a Dkk peptide or LRP polypeptide, to induce a dopaminergic fate or activate β-catenin in stem, neural stem, precursor or progenitor cells . Thus, the method may screen for a substance which modulates the ability of a Dkk ligand or Dkk receptor, such as a Dkk peptide or LRP polypeptide, to induce proliferation, self renewal, dopaminergic development, differentiation, maturation and/or acquisition of a dopaminergic fate in stem, neural stem, precursor, progenitor or neural cells.

Such a method may include:

(i) providing stem, embryonic stem, neural stem, progenitor, precursor or neural cells in the presence of a Dkk ligand and/or Dkk receptor, such as a Dkk peptide and/or a LRP polypeptide and one or more test substances;

(ii) analysing the proportion of such cells which adopt a dopaminergic fate or phenotype and/or respond to the Dkk ligand and/or Dkk receptor; and;

(iii) comparing the proportion of such cells which adopt a dopaminergic fate with the number of such cells which adopt a dopaminergic fate or phenotype and/or respond to a Dkk ligand and/or Dkk receptor in comparable reaction medium and conditions in the absence of the test substance or test substances. A difference in the proportion of dopaminergic neurons between the treated and untreated cells is indicative of a modulating effect of the relevant test substance or test substances

Such a method of screening may include:

(i) treating stem, embryonic stem, neural stem, precursor or progenitor cells or neuronal cells with a Dkk ligand and/or Dkk receptor, such as a Dkk ligand and/or LRP polypeptide, in the presence of one or more test substances; (ii) analysing the proportion of stem, embryonic stem, neural stem, precursor or progenitor cells or neuronal cells which adopt a dopaminergic fate or phenotype and/or respond to the Dkk ligand and/or Dkk receptor; (iii) comparing the proportion of stem, embryonic stem, neural stem, precursor, progenitor or neural cells which adopt a dopaminergic fate or phenotype and/or respond to the Dkk ligand and/or Dkk receptor with the number of stem, precursor, progenitor or neural cells which adopt a dopaminergic fate or phenotype and/or respond to the Dkk ligand and/or Dkk receptor in comparable reaction conditions in the absence of the test substance or test substances.

In other embodiments, methods of screening may include: (i) treating stem, embryonic stem, neural stem, precursor or progenitor cells or neuronal cells with a Dkk ligand and/or Dkk receptor, such as a Dkk ligand and/or LRP polypeptide, in the presence of one or more test substances;

(ii) analysing the proportion of stem, embryonic stem, neural stem, precursor or progenitor cells or neuronal cells which undergo β- catenin activation in response to the Dkk ligand and/or Dkk receptor;

(iii) comparing the proportion of stem, embryonic stem, neural stem, precursor, progenitor or neural cells which cells which undergo β- catenin activation in response to the Dkk ligand and/or Dkk receptor with the number of stem, precursor, progenitor or neural cells which undergo β-catenin activation in comparable reaction conditions in the absence of the test substance or test substances .

Such screening methods may be carried out on cells in vivo in comparable or identical non-human animals, or in vitro or in culture.

As described above, substances screened in accordance with the present invention may be natural or synthetic chemical compounds, including, for example, polypeptides, nucleic acids and small organic molecules.

In other embodiments, a method of screening for a compound useful in inducing or promoting dopaminergic neuronal development by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and/or maturation in a neural stem, embryonic stem, progenitor or precursor cell, may comprise; determining the ability of a proprotein convertase to proteolytically cleave a Dkk polypeptide in the presence of a test compound.

An increase or decrease in said ability in the presence relative to the absence of the test compound is indicative that the test compound is useful in inducing or promoting dopaminergic neuronal development.

The ability of a proprotein convertase to proteolytically cleave a Dkk polypeptide may determined by determining Dkk activity, for example by measuring or detecting β-catenin activation or doperminergic development, or by determining the presence of products of proteolytic cleavage of the Dkk polypeptide.

Suitable Dkk polypeptides include Dkk-1, Dkk-2, Dkk-3 and Dkk-4.

The proprotein convertase may be a protease which is specific for the sequence Arg-X-X-Arg, Arg/Lys-X-X-X-Arg/Lys-Arg, or Arg/Lys-Arg, such as furin or protease convertase 2 (PC2) .

In other embodiments, a method of screening for a Dkk ligand which is not susceptible to proteolysis may comprise; determining the Dkk activity of sample suspected of containing the Dkk ligand in the presence of a proprotein convertase, the presence of activity being indicative that the sample contains said Dkk ligand.

The Dkk activity of the sample may be determined by determining the effect of said sample on promoting dopaminergic neuronal development by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and/or maturation in a neural stem, embryonic stem, progenitor or precursor cell, by determining the effect of said sample on β- catenin activation in a neural stem, embryonic stem, progenitor or precursor cell or by determining the presence or absence of products of proteolytic cleavage of the Dkk ligand.

A method may comprise identifying a sample which contains a Dkk ligand which is not susceptible to proteolysis. The Dkk ligand may be isolated and/or purified from the sample.

A screening method may include purifying and/or isolating a substance or substances from a mixture or sample. The method may include determining the ability of one or more fractions of the mixture to interact with a stem cell, neural stem cell or neural progenitor or precursor cell or neural cell in the presence of Dkk ligand such as a Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide, e.g. the ability to bind to and/or promote the proliferation and/or self-renewal and/or enhance induction, acquisition, differentiation or development of a dopaminergic phenotype and/or neurotransmission or fate in such a stem, neural stem, precursor, progenitor or neural cell. The purifying and/or isolating may employ any method known to those skilled in the art.

A screening method may employ an inducible promoter operably linked to nucleic acid encoding a test substance. Such a construct is incorporated into a host cell and one or more properties of that cell under the permissive and non-permissive conditions of the promoter are determined and compared. The property determined may be the ability of the host cell to induce a dopaminergic phenotype or β catenin activation in a stem, neural stem, precursor, progenitor or neural cell in the presence of Dkk ligand such as a

Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide. A difference in that ability of the host cell between the permissive and non-permissive conditions indicates that the test substance may be able, either alone or in combination, to enhance proliferation and/or self-renewal and/or induction of a dopaminergic fate and/or dopaminergic differentiation or neurotransmission, survival or development in a stem, neural stem or progenitor or precursor cell or enhance dopaminergic induction or neurotransmission or differentiation in a neuronal cell in the presence of Dkk ligand and/or Dkk receptor.

Those of skill in the art may vary the precise format of any of the screening methods of the present invention using routine skill and knowledge. Any of substance tested for its modulating activity may be a natural or synthetic chemical compound.

A compound, substance, factor or factors identified by any one of the methods provided by the invention may be isolated and/or purified and/or further investigated. It may be manufactured and/or used in the preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug.

In various further aspects, the invention further provides a factor identified by any one of the methods disclosed herein, a pharmaceutical composition, medicament, drug or other composition comprising such a factor (which composition may include a stem, neural stem or progenitor or precursor cell or neuron along with a Dkk ligand such as a Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide) , use of such a factor to enhance induction and/or phenotypic differentiation or maturation and/or survival and/or neuritogenesis and/or synaptogenesis and/or neurotransmission and/or functional output of dopaminergic neurons derived from stem, neural stem, embryonic stem or progenitor or precursor cells in the presence of a Dkk ligand such as a Dkk polypeptide and/or a Dkk receptor, such as a LRP polypeptide, use of such a factor or composition in a method of medical treatment, a method comprising administration of such a factor or composition to a patient, e.g. for treatment (which may include preventative treatment) of a medical condition associated with degeneration, damage to, loss of, or a disorder in or affecting dopaminergic neurons, e.g. for treatment of Parkinson's disease or another neurodegenerative disease, use of such a factor in the manufacture of a composition, medicament or drug for administration, e.g. for treatment of Parkinson's disease or other (e.g. neurodegenerative diseases), and a method of making a pharmaceutical composition comprising admixing such a factor with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety.

The invention encompasses each and every combination and sub- combination of the features that are described above.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below.

Figures 1 to 6 show the sequential expression of Dkkl, -2 and -3 in the developing VM. Statistical analysis was performed with one-way ANOVA with Fisher's post hoc test.

Figure 1 shows real time RT-PCR analysis indicating that Dkk-1 is expressed at high levels at early stages in the ventral midbrain.

Figure 2 shows real time RT-PCR analysis indicating that Dkk-2 has a peak of expression by Ell.5.

Figure 3 shows real time RT-PCR analysis indicating that Dkk-3 has a peak of expression at stages after Ell.5. Figure 4 shows Sagittal sections of mouse embryos at Ell.5 and E15.5 were hybridised with TH (a,e), Dkkl (b,f) and Dkk2 (c,g) mRNA probes. At Ell.5, TH is expressed in the DA neurons in the mesencephalic flexure (a) . Dkkl is also expressed in the same region (b, ) and in the diencephalon. Dkk2 is expressed in the marginal and ventral zones of the mesencephalic flexure (c) . At E15.5, TH is expressed in higher levels in the VM, while Dkk-1 and Dkk-2 expression is greatly reduced; Legend: te, telencephalon; ov, optic vesicle; di, diencephalon; mes, mesencephalon,- hb, hindbrain; ex - cortex; cp - cerebellum primordium; rmd - roof of midbrain.

Figure 5 shows that Pl VM glia (VM G) expressed more Dkk-2 (B) than glia from Pl cortex (CxG) or striatum (Str G) , as assessed by realtime PCR.

Figure 6 shows that Pl VM glia (VM G) expressed higher levels of Dkk-3 (B) than glia from Pl cortex (CxG) or striatum (Str G) , as assessed by real-time PCR. Dkk-1 is not expressed by any of the glias.

Figure 7 shows that treatment of E14.5 VM precursors for 3 days with increasing concentrations of partially purified conditioned media (p.p. cm.) from HEK 293T cells over-expressing Dkk-2-AP significantly increased the number of TH + cells, as assessed by immunocytochemistry. Instead, Dkkl did not have any significant effect. Statistical analysis was performed using one-way ANOVA with Fisher's pos-hoc test

Figure 8 shows the number of Nurrl immunoreactive cells was unaffected by the treatment of VM precursors with different Dkks-AP. Statistical analysis was performed using one-way ANOVA with Fisher's pos-hoc test.

Figure 9 shows the treatment of E14.5 VM precursors for 3 days with increasing concentrations of Dkk-2-AP increased significantly the percentage of Nurrl precursors acquiring a TH positive phenotype, while Dkk-3-AP had a significant effect only at 2.5μl/ml. Dkk-l-AP had no effect. Statistical analysis was performed using one-way ANOVA with Fisher's pos-hoc test.

Figure 10 shows the treatment of E14.5 VM precursors for 3 days with increasing concentrations of Dkk-2-AP increased significantly the percentage of Nurrl precursors acquiring a TH positive phenotype, as assessed by double immunocytochemistry .

Figure 11 shows that treatment of E14.5 VM precursors for 3 days with 5-10ng/ml recombinant Dkk-1 decreased the percentage of TH immunoreactive cells out of the total cell population(assessed by Hoechst 33258 staining) , when compared to control (no treatment) . Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test.

Figure 12 shows treatment of E14.5 VM precursors with 5-10ng/ml recombinant Dkk-1 decreased the percentage of TH immunoreactive cells out of the total cell population (assessed by Hoechst 33258 staining) , when compared to control (Ong/ml) .

Figure 13 shows that treatment of E14.5 VM precursors for 3 days with 50-100ng/ml recombinant Dkk-2 decreased the percentage of TH immunoreactive cells out of the total cell population(assessed by Hoechst 33258 staining) , when compared to control (no treatment) . Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test.

Figure 14 shows that treatment of E14.5 VM precursors for 3 days with 50-100ng/ml recombinant Dkk-2 decreased the percentage of TH immunoreactive cells out of the total cell population(assessed by Hoechst 33258 staining) , when compared to control (no treatment) . Figure 15 shows the sequence of human and mouse Dkk-1 and PC2 cleavage fragments. Mouse and human Dkk-1 contain potential minimal furin cleavage sites (Arg-X-X-Arg) , acidic pH furin cleavage sites (Arg/Lys-X-X-X-Arg/Lys-Arg) or protease convertase 2 (PC2) cleavage sites (Arg/Lys-Arg) (Rockwell et al. , 2002; Thomas, 2002) in the middle of their first cystein rich domain (CRD-I) . In addition, Dkk- 1 has potential PC2 cleavage sites in the spacer region between CRD- 1 and the second rich domain (CRD-2) , and also within CRD-2 . According to these putative cleavage sites, upon proteolysis Dkk-1 could be cleaved into 4 fragments (bottom panel) , rendering inactive both the first cystein rich domain (CRD-I) and the second (CRD-2) .

Figure 16 shows the sequence of human and mouse Dkk-2 and PC2 cleavage fragments. Mouse and human Dkk-2 contain potential minimal furin cleavage sites (Arg-X-X-Arg) , acidic pH furin cleavage sites (Arg/Lys-X-X-X-Arg/Lys-Arg) or protease convertase 2 (PC2) cleavage sites (Arg/Lys-Arg) (Rockwell et al . , 2002; Thomas, 2002) in the middle of their first cystein rich domain (CRD-I) . According to these putative cleavage sites, Dkk-2 would be cleaved into 2 fragments, one of which containing a functional CRD-2 (bottom panel) .

Figure 17 shows proteolysis of Dkks modulates their effects in DA neurogenesis .

Figure 17A shows P.p. cm. from HEK 293T cells over-expressing Dkk- 1-AP and Dkk-2-AP contained two bands with high (full length - -105- HOkDa) and low molecular weights (arrow and arrowhead, ~80-85kDa) , which are recognized by a human AP antibody, as assessed by western blotting. The AP tag has a molecular weight of ~67kDa. Equal amounts of protein were loaded.

Figure 17B shows treatment of HEK 293T cells over-expressing FLAG tagged Dkks, with the general proprotein convertase inhibitor decanoyl-RVKR-CMK for 1 day, led to an increase of the secreted full length, unproteolysed fractions of Dkk-1 and Dkk-2, as assessed by- western blotting of the cm. with an anti-FLAG antibody.

Figure 17C shows P.p. cm. from SN4741 cells over-expressing FLAG tagged Dkks, as assessed by western blotting with an anti-FLAG antibody. Lysates from full length Dkk-1-FLAG showed two bands, corresponding to the full-length protein (~4OkDa) and a proteolysed fragment (~25kDa, arrowhead) . Full-length Dkk-2-FLAG was apparently fully proteolysed, 27 as only two bands below 25kDa were detected (arrows) .

Figure 18 shows proteolysis of Dkks modulates their effects in DA neurogenesis. Treatment of E14.5 VM precursors with 2.5μl/ml of p.p. cm. containing Dkk-l-CRD-1 (Dkk-l-Cl) or Dkk-2-CRD-l (Dkk2-Cl) increased the percentage of TH immunoreactive cells out of the total cell population (assessed by Hoechst 33258 staining) , when compared to control p.p. cm.. Scale bar 50μm. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test.

Figure 19 shows that treatment of E14.5 VM precursors with 2,5μl/ml of p.p.cm. containing Dkk-l-CRD-1 (Dkk-l-Cl) , Dkk-2-CRD-l (Dkk2-Cl) or Dkk-2-CRD-2 (Dkk-2-C2) increased the percentage of TH immunoreactive cells out of the total cell population (assessed by Hoechst 33258 staining), when compared to control p.p. cm. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test.

Figure 20 shows proteolysis of Dkks modulates their effects in DA neurogenesis. Treatment of E14.5 VM precursors with 2.5μl/ml of p.p. cm. containing Dkk-2-CRD-2 (Dkk-2-C2) increased the percentage of TH immunoreactive cells out of the total cell population (assessed by Hoechst 33258 staining), when compared to control p.p. cm.. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test. Figure 21 shows that recombinant Dkk-1 (A) or Dkk-2 (B) inhibits basal and Wnt-3a mediated canonical Wnt signalling in SN4741 cells , as assessed by western blot with an antibody against active (5- catenin. (C) P.p. cm. containing Dkk-2 or Dkk-2-CRD-2 (lOμl/ml) synergize the basal canonical Wnt signalling in SN4741 cells, upon co-treatment with Wnt-3a, as assessed by western blot with an antibody against active β-catenin.

Figure 22 shows real-time RT-PCR analysis indicating that LRP-5 is expressed at moderate levels in the ventral midbrain.

Figure 23 shows real-time RT-PCR analysis indicating that LRP-6 has a peak of expression by Ell.5.

Figure 24 shows real-time RT-PCR analysis indicating that Kremen-1 has a peak of expression by Ell.5. Kremen-2 is not expressed.

Figure 25 shows sagittal sections of mouse embryos at Ell.5 were hybridised with LRP-5 and LRP-6 mRNA probes for in situ hybridisation. Both genes are ubiquitously expressed in all brain structures, including VM region with TH+ DA neurons. Legend: te, telencephalon; ov, optic vesicle; di, diencephalon; mes, mesencephalon; hb, hindbrain;

Figure 26 shows that recombinant Kremen-1 does not affect the percentage of TH positive cells from E14.5 VM precursors, when compared to control (no treatment) . Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test. Concentrations in ng/ml .

Figure 27 shows that recombinant Kremen-2 does not affect the percentage of TH positive cells from E14.5 VM precursors, when compared to control (no treatment) . Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test. Concentrations in ng/ml .

Figure 28A to 28C shows that LRP-6N-FC significantly increases the DA differentiation of E14.5 VM precursors. Figure 28A shows treatment of E14.5 VM precursors for 3 days with increasing concentrations of LRP-6N-Fc increased significantly the number of tyrosine hydroxylase (TH) positive cells, as assessed by immunocytochemistry. Figure 28B shows that the number of Nurrl positive cells was unaffected by the treatment of VM precursors with LRP-GN-Fc. Figure 28C shows treatment of VM precursors with LRP-6N- Fc increased significantly the percentage of Nurrl precursors acquiring a TH positive phenotype. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test.

Figure 29 shows expression of the midbrain patterning genes such as En-I, Fgf-8 and Shh was not altered in the VM of LRP-6 homozygote mutant mice at E12.5, when compared to control.

Figure 30 shows the proliferative capacity of Ell.5 VM precursors was not affected in LRP-6 homozygote mutant mice , as assessed by immunohistochemistry against BrdU at ElO.5

Figure 31 shows that the proliferative capacity of Ell.5 VM precursors was not affected in the LRP-6 homozygote mutant mice, as assessed by cell counting with anti-phospho-histone-3 antibody (n=3) Statistical analysis was performed using paired t-test.

Figure 32 shows that the proliferative capacity of Ell.5 VM precursors was not affected in the LRP-6 homozygote mutant mice, as assessed by immunohistochemistry with anti-phospho-histone-3 antibody. Figure 33 shows that the expression of nestin in Ell.5 VM precursors was not affected in the LRP-6 homozygote mutant mice, as assessed by- real time RT-PCR analysis of LRP-6 homozygote mutant VM with primers for the progenitor marker nestin (n=4) . Statistical analysis was performed using paired t-test.

Figure 34 shows that the expression of nestin in Ell.5 VM precursors was not affected in the LRP-6 homozygote mutant mice, as assessed by immunohistochemistry with an antibody against the progenitor marker nestin

Figure 35 shows that the expression of AHD2 in Ell.5 VM precursors was not affected in the LRP-6 homozygote mutant mice, as assessed by real time RT-PCR analysis of LRP-6 homozygote mutant VM with primers for the DA progenitor marker AHD-2. Statistical analysis was performed using paired t-test.

Figure 36 shows coronal sections through Ell.5 VM revealed reduced number of DA neurons (as assessed by TH immunostaining) in the LRP-6 homozygote mutant mice, when compared to control. This reduction took place mainly at the expense of DA neurons in the midline region. General neuronal differentiation (as assessed by β tubulin III (TUJ)) was not affected, although only fibers, but not cell bodies were found in the marginal zone at the midline level.

Figure 37 shows counts of the total number of TH+ cells in the Ell.5 VM (at all levels) in LRP-6 mutant mice (as assessed by TH immunostaining) , indicating a 50% reduction in the LRP-6 mutant mice, when compared to control (n=3) . Neuronal differentiation (as assessed by β tubulin III (TUJ) ) was apparently not affected. Statistical analysis was performed using paired t-test. Figure 38 shows counts of the total number of TH+ cells in the Ell.5 VM (at all levels) in LRP-6 mutant mice as assessed by real time RT- PCR, and indicates a similar decrease in the mRNA levels of TH in the VM of LRP-6 homozygote mutant mice (n=6) . Statistical analysis was performed using paired t-test.

Figure 39 shows in situ hybridisation with TH probe showed decreased levels of TH mRNA in the VM of LRP-6 homozygous mutant mice at the Ell.5.

Figure 40 shows coronal sections through E13.5 VM revealed a partial recovery in the number of DA neurons in the LRP-6 homozygote mutant mice, when compared to stage Ell.5.

Figure 41 shows counts of the total number of TH+ cells in the E13.5 VM (at all levels) in LRP-6 mutant mice (as assessed by TH immunostaining) , indicating a 25% reduction in the LRP-6 mutant mice, when compared to control (n=4) . Statistical analysis was performed using paired t-test.

Figure 42 shows that mRNA levels of TH in the VM were not significantly reduced at E13.5 in the LRP-6 homozygote mutant mice (n=3) . Statistical analysis was performed using paired t-test.

Figure 43 shows immunostaining results showing that at E17.5 the number of DA neurons was fully recovered in the substantia nigra (SN) and ventral tegmental area (VTA) nuclei in the LRP-6 homozygote mutant VM.

Figure 44 shows sagittal sections assessed by in situ hybridisation which indicate that LRP-5 mRNA were increased in the homozygote mutant LRP-6 VM at E15.5, while LRP-6 mRNA could not be detected.

Figure 45 shows coronal sections through Ell.5 VM revealed that the number of Nurrl÷ precursors was reduced in the LRP-6 homozygote mutant mice, when compared to control. Figure 46 shows counting of the total number of Nurr-1+ cells in the Ell.5 VM (at all levels) indicated a reduction in the LRP-6 homozygote mutant mice, when compared to control (n=3) . Statistical analysis was performed using paired t-test.

Figure 47 shows real time RT-PCR analysis of VMs from LRP-6 homozygote mutant mice revealed a similar decrease in the Nurr-1 mRNA levels (n=6) . Statistical analysis was performed using paired t-test.

Figurer 48 shows immunostaining against Nurr-1 in coronal sections through E13.5 VM and real time RT-PCR revealing that there was no reduction in the number of Nurr-1+ progenitors at this stage in the LRP-6 homozygote mutant mice, when compared to control (n=4) .

Figure 49 shows real time RT-PCR analysis of VMs from LRP-6 homozygote mutant mice at Ell.5 revealed a decrease in the mRNA levels of Pitx-3 (n=5) . Statistical analysis was performed using paired t-test.

Figure 50 shows In situ hybridisation for the DA differentiation marker Pitx-3 showed decreased expression in the VM at E12.5 VM.

Figure 51 shows no reduction in the number of Nurr-1+ progenitors at this stage in the VM of the LRP-6 homozygote mutant mice at E13,5, when compared to control (n=4) . Statistical analysis was performed using paired t-test.

EXPERIMENTAL Specifically incorporated by reference herein are the experimental results set out in WO00/66713 demonstrating proliferation and/or self-renewal of dopaminergic precursors and induction of dopaminergic neurons in stem, neural stem, precursor or progenitor cells expressing Nurrl, in the presence of type 1 astrocytes or glial cells, and demonstrating additional results obtained when contacting such cells with additional factors, such as FGFs (e.g. FGF8) or retinoids and WO2004/029229 demonstrating proliferation and/or self-renewal of dopaminergic precursors and induction of dopaminergic neurons in stem, neural stem, precursor or progenitor cells by expressing a nuclear receptor of the NG4A subfamily, e.g. Nurrl, above basal levels within the cell and treating the cell with a Wnt ligand.

Partial purification of conditioned media containing Dkks-AP and Dkks-FLAG and furin inhibitor treatments

Human embryonic kidney 293T (HEK 293T) or SN4741 cells were transiently transfected with Dkks-AP (Mao et al. , 2001a) or full- length and truncated Dkks-FLAG (Li et al . , 2002) plasmids, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48 hours, conditioned serum-free N2 media was collected and concentrated with Centricon-Plus 20 columns (Millipore) according to the manufacturer's instructions. Protein content was measured and aliquots with equivalent concentration were prepared and stored at -8O⁰C. In the furin inhibition experiments, DMSO (as a control) or decanoyl-RVKR-CMK (Calbiochem) was added to the cultures 4 hours after the beginning of the transfection, when the OPTIMEM media (Invitrogen) was replaced by N2 media; cm. was collected after 24 hours, centrifugated, to remove any cell contamination, and directly analysed (without p.p.) .

Precursor cultures and treatments

Embryonic day (E) 14.5 VM obtained from time-mated Sprague-Dawley rats (ethical approval for animal experimentation was granted by Stockholms Norra Djurfδrsδks Etiska Natnnd) were dissected, mechanically dissociated, and plated at a final density of IxIO⁵ cells per cm² on poly-D-lysine (lOμg/ml) coated plates (Falcon) in serum-free N2 medium, consisting of a 1:1 mixture of F12 and MEM (Invitrogen) with 15mM HEPES buffer and ImM glutamine (Invitrogen) . N2 supplement and 5mg/ml Albumax (from Invitrogen) were also added to the media, or, in alternative, 5μg/ml insulin, lOOμg/ml apo- transferrin, 60μM putrescine, 2OnM progesterone, 3OnM selenium, 6mg/ml glucose, and lmg/ml BSA (all purchased from Sigma) . Cultures were grown three days in vitro, in a 37⁰C incubator, at 5% CO₂, before fixation. Recombinant mouse Dkk-1 (R&D Systems) and recombinant human Dkk2 (AmProx, USA) were used at a final concentration of 0-500 ng/ml. Dkks-AP and truncated Dkks-FLAG were added at 0-10μl/ml of a 25μg/μl stock solution. All these reagents were added to the precursor cultures at the beginning of the cultures .

Dkks-AP and LRP-6N-Fc were added at 0-10μl/ml of a 25μg/μl stock solution. All these reagents were added to the precursor cultures at the beginning of the cultures.

Glial cultures

Glial cultures (O'Malley et al . , 1992) were established from postnatal day 1 (Pl) VM, cortex (Cx) and striatum (STR) , obtained from time-mated Sprague-Dawley rats. Following mechanical dissociation, 10xl0^s cells were plated in previously lOμM poly- D-lysine coated 25cm² flasks, in NM15 medium (containing 15% fetal bovine serum, 7.2mg/ml glucose, 2.4mM glutamine, 0.5% Fungizone and 0.6% gentamycin in MEM (all from Invitrogen) ) . Cells were grown to monolayer and microglia and debris were removed by shaking for 1 hour at 37⁰C and 190 rpm. The attached cells were then washed with sterile PBS and incubated for 1-2 hours in a 37⁰C incubator, at 5% CO₂, before overnight shaking at 37^CC and 210 rpm. The supernatant media containing oligodendrocyte precursors and other cell types was then discarded. The remaining glial monolayer was then lysed for RNA collection.

Real-time PCR and quantification of gene expression

Genbank cDNA sequences were used to design primers in Primer Express 2.0 (PE Applied Biosystems,Foster City, CA, USA) . The specificity of PCR primers was determined by BLAST run of the primer sequences. The oligonucleotides set out in Table 1 were used. Apart from Quantum RNA classical 18S internal standard (Ambion, Austin, USA) , all primers were purchased from DNA Technologies, Denmark.

Total RNA was isolated from pools of ventral mesencephalon dissected from ElO.5, Ell.5, E13.5, E15.5, and Pl rats, and from glial monolayers (n=4, for each region), using Rneasy extraction kit (Qiagen,Hilden, Germany) . For reverse transcription reactions, 1 μg of total RNA was initially treated with 1 unit RQl Rnase-free DNAse (Promega, Madison, USA) for 40 minutes. The DNAse was inactivated by the addition of lμl of EDTA 0.02M and incubation at 65⁰C for 10 minutes. 0.5 μg random primers (Invitrogen) were then added, and the mixture was incubated at 65°C for 5 minutes. Each sample was then equally divided in two tubes, a cDNA reaction tube and a negative control tube. A master mix containing Ix First-Strand Buffer (Invitrogen), 0.0IM DTT (Invitrogen) and 0.5mM dNTPS (Promega, Madison, USA) was then added to both cDNA and RT- tubes and incubated at 25⁰C for 10 minutes, followed by a 2 minutes incubation at 42°C. Supercript II reverse transcriptase (200 units, Invitrogen) was then added only to the cDNA tubes and all samples were incubated at 42°C for 50 minutes. Superscript II was inactivated by incubation for 10 minutes at 70^CC. Both cDNA and RT- were then diluted 10 times, for further analysis.

Real-time RT-PCR reactions were performed in a triplicate master mix, with lμl 10x diluted cDNA or RT-/25 μl final volume. Each PCR reaction consisted of Ix PCR buffer (Invitrogen) , 3mM MgCl₂ (Invitrogen), 0.2mM dNTPs (Promega, Madison, USA), 0.3μM of each of the forward and reverse primers, 0.5 unit Platinum Taq DNA polymerase (Invitrogen) and Ix SYBR Green (Molecular Probes, Leiden, The Netherlands) . The PCR was performed at 94⁰C for 2 minutes and then for 35-40 cycles at 94° C for 30 s, at 60⁰C for 30s, at 72° C for 15s and at 80⁰C for 5s (forSYBR Green detection) on the ABI PRISM 5700 Detection System (PE Applied Biosystems, Foster City, CA, USA) . A melting curve was obtained for each PCR product after each run, in order to confirm that the SYBR Green signal corresponded to a unique and specific amplicon. Standard curves were generated in every 96-well plate for every real time PCR run, using serial 3-fold dilutions of a control sample containing the sequence of interest (reverse transcribed RNA, plasmid containing or genomic DNA) for every probe. The resulting standard curve plots were then used to convert the Cts (number of PCR cycles needed for a given template to be amplified to an established fluorescence threshold) into arbitrary quantities of initial template of a given sample. The expression levels were obtained by subtracting the RT- value for each sample from the corresponding RT+ value (when appropriate) and then dividing that number by the value of the house-keeping gene,18S, obtained for every sample in parallel assays. Each assay for a particular gene was repeated twice. 18S assays were run for each sample, at the beginning and once or twice in the middle of assays, to verify the integrity of the samples.

PCR products were run in a 2% agarose gel to verify the size of the amplicon. Statistical analysis of the results was performed by oneway ANOVA. Fisher's protected least significant difference was used post hoc to identify specific points at which the different developmental stages differed from the earliest, only when significant interactions occurred. Significance for all tests was assumed at the level of p<0.05 (*p<0,05; ** p<0,001; *** p<0,0001) .

In situ hybridisation

Wild type and Lrpβ mutant mouse embryos at Ell.5, E12.5 and E15.5 were fixed overnight in 4% paraformaldehyde at 4⁰C and embedded in paraffin. Serial sections of 8 μrα were processed for radioactive in situ hybridisation using [S35] -UTP labelled antisense riboprobes. Hybridisation was carried out at 56⁰C in 50% formamide according to a modified protocol of Dagerlind et al . (Dagerlind et al . , 1992) . Sections were counterstained with Cresyl Violet (Sigma) . Probes for in situ hybridisation were as follows: Dkkl, Dkk2 (Monaghan et al. , 1999) , Lrp5, Lrp6 (PCR-products for regions 2884-3444bp for Lrp5 NM_008513 and 2941-3446bp for Lrpδ NM_008514) , TH, Pitx3 (kindly- given by Jordi Guimera, (Brodski et al . , 2003)), EnI (Davis and Joyner, 1988), Fgf8 (Martinez et al. (1999) and Shh.

BrdU immunostaining

For proliferation assay BrdU Detection kit II (Roche, Germany) was used with slight modifications. Pregnant mice were injected peritoneally with 5-bromo-2-deoxyuridine (BrdU, lOOOμg/lOOg) 2 hours before sacrificing. Embryos/brains were incubated overnight in 4% paraformaldehyde at 4⁰C, dehydrated through ethanol and rotihistol and paraffin embedded. Paraffin sections (8μm) were deparaffinised in rotihistol, rehydrated, cooked in sodium citrate (0.01 M) for 5 minutes, washed with PBS and incubated 1 hour in blocking solution

(PBS with 10% fetal calf serum, 0.05% Triton XlOO) . Next slides were incubated overnight at 4°C with the first anti-BrdU antibodies (dilution 1:10 in PBS with 0.05% Triton XlOO) . Following three washes with PBS, sections were incubated two hours with the second anti-mouse biotinylated antibodies (1:500, Jackson ImmunoResearch, USA) . Following three washes in PBS, slides were incubated 30 minutes with ABC solution (ABC-kit, Vectastain, Vector Laboratories, USA) and proceeded diaminobenzidine staining (DAB, Sigma) until a signal can bee seen. Slides were washed twice with PBS, dehydrated and lid with Roti-Histokit (ROTH, Germany) .

Immunocytochemistry

Cells were fixed in ice-cold 4% paraformaldehyde for 20 minutes and washed in PBS. They were then blocked by a 15-minute incubation with PBT (PBS with 1% BSA, 0.3% Triton XlOO and 0.02% sodium azide, all from Sigma) . After an overnight incubation at 4⁰C with the primary antibody (mouse anti-TH (1:1000 dilution - Immunostar) , rabbit anti- TH (1:250 - Pelfreeze) rabbit anti-Nurrl (1:1000 - Santa Cruz)) , the cells were washed twice with PBS, for 10 minutes and incubated for 2 hours with an appropriate secondary antibody (biotinylated 1:500 -α- mouse or α-rabbit IgG (Vector) , Cyanine-2 or rhodamine-coupled horse-α-mouse IgG 1:200 or goat α-rabbit IgG 1:200 (Jackson Laboratories)) . After 2 washes with PBS, the cells were either counterstained with Hoechst 33258 for 10 minutes (in case of immunofluorescence) or incubated with avidin-biotin complex solution (Vector) for 2 hours. After 2 washes with PBS, the staining was developed using chromogenic VIP Vector Laboratory ABC immunoperoxidase kit (red staining - TH) or 3-3' diaminobenzidine tetrahydrochloride (DAB 0.5mg/ml) /nickel chloride (1.6mg/ml) (gray/black - Nurrl) .

Nurrl staining was done prior to TH staining. Images were acquired from stained cells in PBS at room temperature with a Zeiss Axioplan IOOM microscope (LD Achrostigmat 2Ox, 0.3 PHl ∞ 0-2 and LD Achroplan 40x, 0,60 Korr PH2 ∞ 0-2) and collected with a Hamamatsu camera C4742-95 (with QED imaging software) . Images were processed with Adobe Photoshop version 7.0.

Quantitative immunocytochemical data represent means + standard error of the mean, obtained from 10 non-overlapping fields in 2-3 replicates per condition from 2-3 separate experiments. Statistical analysis was performed in Stat View (Cary, NC, USA) or Prism 4 software (Graph Pad, San Diego, USA) as described in the figure legends and significance for all tests was assumed at the level of p<0.05 (* p<0,05; ** 0, 01<p<0, 001; *** p<0,001) .

Immunoblotting

Partially purified conditioned media from Dkk-AP and LRP-6N-Fc overexpressing HEK 293 cells were mixed with Ix Laemmli buffer (1:20) . The samples were boiled and subjected to 10% SDS PAGE (15μl of media sample per lane) and electrotransferred onto Hybond-P PVDF membrane.

Membranes were blocked in 5% milk and immunodetected using anti- human placental alkaline phosphatase (PLAP, Abeam) , anti-Dkk-3 (R&D Systems) and anti-human IgG (Vector) specific primary antibodies and appropriate secondary antibody. Signal was visualized by ΞCL+Plus detection system (Amersham Biosciences) according to manufacturer's instructions. After immunodetection, total protein was stained by amidoblack to confirm equal protein loading.

Iimunohistochemistry for LRP-6 mutants

LRP-6 mutants (LRP-6-/-) (Pinson et al. , 2000) were housed, bred, and treated in accordance with the ethical approval for animal experimentation granted by Stockholms Norra Djurfδrsδks Etiska Namnd. Wild-type and heterozygous mice were identified with genotyping PCR reactions with the previously described primers LRP- 6-Ul and LRP-6-D1 (Kelly et al . , 2004), while mice with the gene trap insertion were recognized with the following primer set: CD4mix forward: 5'-GCACGGATGTCTCAGATCAAGAGG- 3' and CD4mix reverse: 5'- CGGGATCATCGCTCCCATATATG-S', with a annealing temperature of 63⁰C and an amplicon of 108bp. Ear or embryonic tissues were boiled at 95°C for 40 minutes in 100- 200μl of 25mM NaOH/0,2mM EDTA and an equal volume of 4OmM Tris HCl ph 5 was added. 3μl of this solution was used in PCRs, which were performed as described for the real time-PCR, but without SYBR Green, without the 80°C step, and for 30 cycles.

Ell.5, E13.5 and E17.5 mice were fixed with 4% paraformaldehyde overnight and immersed in a 20% sucrose gradient. Samples were then rapidly frozen in O.C.T at -80⁰C. Serial sagittal and coronal sections (14μm thick) were collected on microscope slides

(StarFrost) and stored at -80°C. For immunohistochemistry, slides were thawed and incubated for 10 minutes with 4% paraformaldehyde. After three 15 minute washes with PBS, the slides were blocked with PBTG (PBT with 5% goat serum) for 30 minutes. Primary antibody (rabbit α-TH (1:250 - Pelfreeze) , rabbit α-Nurrl (1:1000 -Santa Cruz) and rabbit α-active-caspase III (1:100 - Cell Signalling)) in PBTG was incubated at 4°C overnight.

Following 3 washes with PBS, the sections were blocked with PBTG for 30 minutes and incubated for 2 hours with a secondary antibody (Cyanine-2 or rhodamine-coupled horse-α-mouse IgG 1:200 or goat α- rabbit IgG 1:200 (Jackson Laboratories) . Slides were then washed three times with PBS for 15 minutes, counterstained with Hoechst 33258 for 1 minute and mounted in PBS/glycerol (1:4) . Images were acquired at room temperature with a confocal laser scanning microscope (Zeiss 510, argon (488nm) and helium-neon (543 and 633nm) lasers) and Zeiss LSM Viewer software. Images were processed with Adobe Photoshop version 7.0. All the panels were assembled using Adobe Illustrator 9.0. Quantitative immunohistochemical data represent means ± standard error of the mean. All the sections where VM was present were counted for each animal and 3-4 pairs of mice (wild-type and LRP-β mutant) were analysed. Statistical analysis was performed using Prism 4 software (Graph Pad, San Diego, USA) as described with paired t-test and significance was assumed at the level of p<0.05 (* p<0,05; ** 0, 01<p<0, 001; *** p<0,001) .

Results Sequential expression of Dkkl, 2 and 3 in the developing VM

Expression profiles of Dkks were analysed in the developing rat VM, by real time PCR. Dkk-1 was highly expressed at E10.5-E11.5, decreasing at E13.5 (Figure 1) . Dkk-2 mRNA levels, however, peaked at Ell.5 (Figure 2), prior to the onset of DA neurogenesis in the rat (Perrone-Capano et al . , 2000) . Interestingly, this pattern of expression is similar to that previously reported for Wnt-5a, a key regulator for DA differentiation (Castelo-Branco et al . , 2003) . Dkk- 3 mRNA was also present at early stages of VM development, but its expression was much higher at post-neurogenic time points (E15.5 and Pl, Figure 3) . Similar results were observed in the developing mouse VM, by in situ hybridisation. Dkk-1 was highly expressed in the mesencephalic flexure (Figure 4b) , overlaying with the expression of TH (Figure 4a) . In addition, its expression was found in a small domain of the diencephalon. At E15.5, Dkk-1 expression was not observed in the midbrain (Fig. 4f) . At Ell.5, Dkk-2 was expressed from the ventricular to the marginal zone, from the diencephalon and the mesencephalic flexure overlaying the TH expression domain (Figure 4c) . Similar to that of Dkk-1, Dkk-2 mRNA was not found in the midbrain of E15.5 mice (Figure 4g) . We also examined if postnatal VM glia, a source of DA instructive signals (Wagner et al . , 1999) (Castelo-Branco et al . 2005) , expressed Dkks. While Dkk-1 was not expressed in any of the analysed glial populations, we found that Dkk-2 and Dkk-3 were expressed in Pl VM glia (Figure 5 and 6) . Interestingly, Dkk-2 expression was specific to VM glia, as it was not detected in glia from the cortex and striatum (Figure 5) , unlike Dkk-3 (Figure 6) . The dynamic pattern of expression of Dkks in vivo and in the inductive VM postnatal glia provided indication of distinct and non-redundant functions for the different Dkks in the developing VM.

In order to investigate the effects of Dkks in the developing VM, we transiently transfected alkalinephosphatase (AP) tagged Dkks (Mao et al . , 2001a) in human embryonic kidney cell line (HEK 293) . Conditioned media was collected, partial purified and the presence of DKKs-AP was confirmed by immunoblot against AP and Dkk-3. We then treated rat E14.5 VM neuronal precursor cultures with increasing doses of partial purified Dkks-AP and assessed their effect on DA precursor differentiation. Dkk-1 did not affect the number of tyrosine hydroxylase (TH) positive cells in the cultures at any concentration, while Dkk-2 significantly increased the number of DA neurons at 2.5 and 5μl/ml (Figure 7) . None of the Dkks affected the total number of Nurrl positive cells in the cultures at any given doses (Figure 8) .

Partially purified Dkk conditioned media and recombinant Dkks have opposite effects on the differentiation of VM precursors Interestingly, when we assessed the number of DA precursors (Nurrl+/TH-) that converted into Nurrl+/TH+ DA neurons, Dkk-2 displayed a powerful and sustained effect from 2.5 to lOμl/ml and Dkk-3 significantly increased this proportion at 2.5μl/ml (Figure 9 and 10) . Therefore, Dkk-2 appeared to be a strong inducer of VM DA neuron differentiation.

To confirm these results, we treated VM precursor cultures with increasing doses of recombinant full-length mouse Dkk-1 (R&D systems) and human Dkk-2 (Amprox) . We found that at 5-10ng/ml Dkk-1 significantly decreased the number of TH+ cells (Figure 11) . However, at higher doses (30-100ng/ml) , recombinant Dkk-1 was no longer effective at reducing the number of TH+ neurons (Figures 11 and 12) . Surprisingly, we observed that recombinant Dkk-2 could also decrease the numbers of TH+ in VM precursor cultures (Figures 13 and 14) , albeit at higher concentrations then recombinant Dkk-1 (50- 100ng/ml) . Therefore, recombinant full-length Dkks exert distinct effects on DA neurogenesis when compared to Dkks produced in mammalian cells. Taken together, this data indicates that Dkks distinctively regulate the differentiation of E14.5 DA VM precursors, with Dkk-2 being a strong inducer and Dkk-1 an inhibitor of their differentiation into TH+ neurons.

Dkks undergo proteolytic processing in cells derived from the developing VM

Full-length recombinant Dkks are produced in Escherichia coli and thus do not undergo the same post-translational modifications occurring in mammalian cells, including proprotein convertase- mediated proteolysis and glycosylation. Therefore, we examined whether such differences in processing could account for the difference in activity between the full-length Dkks and the p.p. conditioned media (cm.) . Immunoblotting of the p.p. cm. with an antibody against AP revealed that both Dkk-1 and Dkk-2 were present in two forms. Apart from the high molecular weight bands (~105- HOkDa) , corresponding to the AP-tagged (~67kDa) full-length proteins (~35-40kDa) , an additional lower molecular weight band (~80-85kDa) was detected in partially purified Dkk-1 and Dkk-2 cm. Mouse and human Dkk-1 and Dkk-2 contain potential minimal furin cleavage sites (Arg-X-X-Arg) , acidic pH furin cleavage sites (Arg/Lys-X-X-X-Arg/Lys-Arg) or protease convertase 2 (PC2) cleavage sites (Arg/Lys-Arg) (Rockwell et al. , 2002; Thomas, 2002) in the middle of their first cystein rich domain (CRD-I) (figures 15 & 16) . In addition, Dkk-1 has potential PC2 cleavage sites in the spacer region between CRD-I and the second rich domain (CRD-2) , and also within CRD-2. Moreover, in the developing VM, furin and other proprotein convertases have been shown to be highly expressed at the time of DA neurogenesis (Zheng et al. , 1994), providing indication that Dkks might be proteolytically cleaved at this stage.

To determine whether Dkks are proteolytically processed, we over- expressed FLAG-tagged Dkk-1 (-4OkDa) and Dkk-2 (-35 kDa) (Li et al . , 2002) in HEK 293T cells treated with increasing doses of decanoyl- RVKR-CMK, an inhibitor of proprotein convertases, including furin and PC2 (Jean et al . , 1998) . We found that 25μM of this inhibitor was sufficient to increase the total amount of full length (-35- 4OkDa) uncleaved Dkk-1 or Dkk-2 fractions. These data provide indication that the lower molecular band in the p.p. cm. was indeed due to proteolytic processing. Furthermore, we observed that this phenomenon also occurred in the SN4741 DA cell line (Son et al . , 1999) . Upon over-expression of FLAG tagged Dkks, we observed that Dkk-1 was present as a ~40kDa full length form and also as a ~25kDa truncated form and that treatment with decanoyl-RVKR-CMK also increased the total amounts of full-length protein (figure 17) . We also found that expression of Dkk-2 in SN4741 cells gave rise to two truncated forms, of ~22-25kDa, but not the full-length protein. These results provide indication that Dkks undergo distinct proteolytic cleavage events in different mammalian cells, including DA cells. Therefore, we next examined whether cleavage fragments including intact cystein-rich domain of Dkk-1 or Dkk-2 had distinct biological activities. The second cystein-rich-domain of Dkk-2 increases the number of TH positive cells in VM precursor cultures

Dkk-1 and Dkk-2 contain two cystein-rich-domains (CRD) (Krupnik et al . , 1999) and truncated proteins containing the different CRDs have been shown to differentially modulate the activation of canonical Wnt signalling (Brott and Sokol, 2002; Li et al . , 2002) . However, as furin and PC2 cleavage sites are located within the CRD-I of both Dkk-1 and Dkk-2 (Figures 15 & 16), and of the CRD-2 of Dkk-1, the Dkk-2-CRD-2 is likely to be the only truncated form mimicking the normal cleavage products. To determine the activity of the different CRDs of Dkk-1 and Dkk-2 in the development of VM DA neurons,' we overexpressed FLAG-tagged CRDs from Dkk-1 and Dkk-2 (Li et al. , 2002) in SN4741 cells, and generated partially purified conditioned media. The different truncated proteins were expressed at moderate levels and were present at the expected molecular weight (Li et al . , 2002) . Treatment with CRD-I of both Dkk-1 and Dkk-2 led to an increase in the numbers of TH+ cells at 2,5μl/ml (Figures 18 and 19) . While no significant change in the numbers of DA neurons was observed with the CRD-2 of Dkk-1, the CRD-2 of Dkk-2 led to an increase at the dose of 2,5μl/ml (Figure 20) . These results indicate that full-length Dkk-1 and Dkk-2 have a distinct activity on DA differentiation, when compared to their proteolytic fragments or CRDs.

Full length Dkks inhibit canonical Wnt signalling, while Dkk-2-CRD-2 have the opposite effect

We examined how Dkks modulated Wnt signalling in a DA cell line. Recombinant full-length Dkk-1 and recombinant full-length Dkk-2 efficiently inhibited canonical Wnt signalling, as shown by the decreased levels of transcriptionally active β-catenin, in SN4741 cells stimulated with low doses of Wnt-3a (figure 21) . Interestingly, the effects of Dkks were less evident at higher doses of Wnt-3a and Dkk-1 was able to inhibit β-catenin signalling at lower doses than Dkk-2, in accordance to their effects on the VM precursors. On the other hand, treatment of SN4741 cells with Dkk2- CRD-2 or Dkk-2 overexpressed and processed by SN4741 cells led to a synergy in the Wnt-3a-mediated activation of β-catenin, while Dkk-1 or Dkk-l-CRD-2 had no effect. These results are in agreement with previous reports showing activation of canonical Wnt signalling by Dkk-2 or Dkk-2-CRD-2 in LRP-6 over-expressing cell lines (Li et al. , 2002; Liu et al . , 2005) or Xenopus (Brott and Sokol, 2002) . Therefore, our results indicate that the differential activity of Dkk CRDs and full-length proteins regulate canonical Wnt signalling and DA neurogenesis and provide indication that proteolysis plays a key role in determining the function of Dkks.

LRP-5, LRP-6 and Kremen-1, but not Kremen-2, are expressed in the developing VM and LRP-6N-FC potentiates DA differentiation Dkk-1 and -2 have been classically defined as extracellular inhibitors of Wnt/β-catenin signalling (Kawano and Kypta, 2003) and we have previously shown that glycogen synthase kinase (GSK) -3 inhibition/stabilization of β-catenin leads to the differentiation of DA neurons (Castelo-Branco et al. 2004) . In order to regulate Wnt/beta-catenin signalling, Dkks have to bind to either LRP-5 or -6 (Bafico et al. , 2001; Mao et al . , 2001a; Semenov et al . , 2001) .

Therefore we analysed the mRNA expression profile of LRPs during VM development. Both LRP-5 and LRP-6 were expressed in the VM (Figures 22, 23 and 24), and interestingly, LRP-6 peaked at Ell.5 (Figure 23) , as previously observed for Dkk-2 (Figure 2) and Wnt- 5a (Castelo-Branco et al . , 2003) . It has been shown that high levels of LRP-6 can enhance Wnt mediated canonical signaling (Brennan et al. , 2004; Holmen et al . , 2002; Liu et al. , 2003) , titrate the inhibitory effects of Dkk-1 (Bafico et al . , 2001; Mao et al . , 2002; Mao et al . , 2001a) and, together with high levels of Dkk-2, activate Wnt/beta-catenin signaling both in Xenopus (Brott and Sokol, 2002; Mao and Niehrs, 2003) and mammalian cells (Li et al. 2002; Mao and Niehrs, 2003) . This provided indication that Dkk-2 modulates DA differentiation through activation of β-catenin, a process also regulated by a second class of Dkk co receptors, the Kremens . Kremen-1 and -2 proteins can bind to Dkks, forming a ternary complex with LRP-6, which is then internalized, preventing signaling through Wnt/beta-catenin (Mao et al . , 2002) .

However, Dkks are however unable to inhibit Wnt/beta-catenin signalling when endogenous Kremens become limiting or when LRP5/6 levels are high (Davidson et al . , 2002; Mao and Niehrs, 2003) . For instance, absence of Kremen-2 can transform Dkk-2 (but not Dkk-1) from an inhibitor of canonical Wnt signalling to an activator (Mao and Niehrs, 2003) . We found that Kremen-1 was present in the developing VM, peaking at Ell.5 (Figure 25) , a stage where both Dkk- 1 and Dkk-2 are still present in high levels. Kremen-2, however, was barely detectable by real time PCR.

These results are consistence with whole-mount in situ hybridization profiles of the Kremens (Davidson et al. , 2002) , showing expression of Kremen-1 but not Kremen-2 in the early VM. Our results provide indication that both Dkk-1 and -2 can interact with Kremen-1 in the early VM, inhibiting Wnt canonical signalling.

However, the absence of Kremen-2 might also prompt Dkk-2 to activate canonical Wnt-signalling, most likely in different cell types in the DA niche. We therefore examined the effects of the treatment of E14.5 VM precursors cultures with soluble Kremen-1 and -2 (R&D systems) . Although the number of TH+ cells tended to decrease, we did not observe any statistical significant change (Figures 26 and 27) . Membrane attachment of Kremen is essential for its binding to Dkks and their Wnt inhibitory function in 293T cells and Xenopus (Davidson et al. , 2002; Mao et al. , 2002) and this requirement might also be valid for the developing VM. In sum, LRPs and Kremens are expressed in specific patterns in the developing VM and might play a role in DA differentiation.

In order to further assess the relevance of LRP-6 in our system, we used LRP-6N-Fc, a secreted fusion protein containing the extracellular domains of LRP-6 and the Fc domain of the human IgG (Tamai et al . , 2000) . We have overexpressed LRP-δN-Fc in HEK293 cells and confirmed its presence in the conditioned media by immunoblot against human IgG. We then treated our E14.5 VM precursor cultures with increasing doses of LRP-6N-FC and found a potent effect on DA differentiation, with significant increase in the numbers of TH+ neurons (Figure 28A) and the percentage of Nurrl precursors converting to a DA phenotype (Figure 28C) . The total number of Nurrl positive cells was not affected (Figure 28B) , providing indication that the effects were on differentiation and not in Nurrl+ precursor proliferation. LRP-6N-Fc has been described to attenuate Wntl-mediated stabilization of beta-catenin (Brennan et al. , 2004; Gonzalez-Sancho et al . , 2004; Holmen et al . , 2002) , but it can also bind to Dkks (Semenov et al . , 2001) . We found that this fusion protein mimicked the DA effects of Dkk-2 (Figure 2) or Wnt-5a (Castelo-Branco et al . , 2003) on DA differentiation.

LRP-6 homozygote mutant mice do not display patterning, proliferation, or cell death defects in the VM

LRP-6 homozygote mutant mice have been described to display several developmental defects associated with de-regulation of Wnt signalling, including an encephaly, and/or a deletion of the caudal midbrain (Pinson et al . , 2000) . These previous observations were confirmed by the observed loss of dorsal expression of midbrain/hindbrain marker genes, such as Engrailed (En-I) and Fibroblast growth factor 8 {fgf-8) . However, no difference in the expression of EnI, fgf-8, Sonic hedgehog {Shh) or FiZnt-Sa was observed in the VM at Ell.5-12, providing indication that the VM forms normally in these mutants (figure 29) . Therefore, the mid- hindbrain boundary defects in LRP-6 homozygous mutants are associated most likely with the loss of dorsal/caudal midbrain structures .

In agreement with these findings, we did not observe any decrease in the proliferation of VM precursors at the Ell.5, as assessed by immunostaining with the S-phase marker BrdU at ElO.5 and Ell.5 (Figure 30) and the cell cycle marker phospho-histone-3 at Ell.5 (Figures 31 and 32) (Hendzel et al. , 1997)) . Moreover, the number of cells undergoing apoptosis in the wild-type or mutant VM at E13.5 did not change, as assessed by active caspase-3 immunostaining. The expression and distribution of the neural stem/progenitor cell marker nestin (Figure 33 and 34) and the mRNA levels of the DA progenitor cell marker aldehyde dehydrogenase 2 {AHD-2, Figure 35) (Wallen et al . , 1999) were not altered in the mutant VM. These results provided indication that deletion of LRP-6 did not alter the normal patterning, proliferation or cell survival of DA precursors in the VM.

Delayed DA neurogenesis in LRP-6 homozygote mutant mice

We examined whether LRP-6 regulated later aspects of DA development in the VM. By Ell.5, the time of birth of DA neurons, we observed a striking reduction in the number of TH+ cells (50%, Figure 36 and 37) , with no apparent change in the general neuronal population (β tubulin III (TUJ) immunoreactive) (figure 36) . TH mRNA was also significantly lower in the homozygote mutant mice, as assessed by real-time RT-PCR (Figure 38) and in situ hybridisation (Figure 39) . At E13.5, the difference in the number of DA cells between wild-type and mutant was reduced to 25% (Figure 40 and 41) and no difference could be detected by real-time RT-PCR at the mRNA level (Figure 42) . At E17.5, the levels of TH were normal at the two main DA nuclei of the mutant mice, the substantia nigra and the ventral tegmental area (Figure 43) . In addition, DA neurons innervated the striatum in the mutant mice. Interestingly, the apparent recovery on the numbers of DA neurons coincided with an increased expression of LRP-5 in the midbrain of the homozygote mutant mice, as assessed by in situ hybridisation (Figure 44) .

We next examined whether the expression of transcription factors required for the normal development of DA neurons was altered in the LRP-6 homozygote mutant mice. First, we focused on Nurr-1, which is essential for late aspects of VM DA development (Castillo et al . , 1998; Le et al . , 1999; Zetterstrom et al . , 1997), including differentiation (Joseph et al. , 2003) . We found that the number of Nurr-1+ cells (both TH- precursors and TH+ DA neurons) was greatly reduced in the mutant VM at Ell.5 (Figures 45 and 46) . A 40% decrease in Nurr-1 mRNA levels was also observed by real-time RT-PCR at this stage (Figure 47) . However at E13.5 the number of Nurr-1+ cells was no longer reduced in the mutant VM (Figure 48) . Furthermore, the expression of Pitx-3, a transcription factor expressed at early stages of DA differentiation (Smidt et al . , 1997) and required for their maintenance (Hwang et al. , 2003; Maxwell et al . , 2005; Nunes et al . , 2003; Smidt et al . , 2004; van den Munckhof et al . , 2003) , was also greatly reduced both at Ell.5 and at E12.5, as assessed by real-time RT-PCR and in situ hybridisation, respectively (Figures 49 and 50) . However, the expression of both these transcription factors approached normal levels by E13.5

(Figure 51) . Thus, our results show that deletion of LRP-6 results in delayed DA neurogenesis, providing indication that LRP-6 is not absolutely required but it is necessary for the adequate onset of DA neurogenesis in the VM.

Dkks are involved in the regulation of several cellular processes, including programmed cell death in the developing limbs (Grotewold and Ruther, 2002) , proliferation of adult human mesenchymal stem cells (Gregory et al . , 2003) , inhibition of invasion and mobility of osteosarcoma cells (Hoang et al . , 2004) and terminal osteoblast differentiation and mineralized matrix formation (Li et al. , 2005) . Here, we have demonstrated for the first time that (1) Dkk-1 and Dkk-2 are key regulators of DA differentiation; (2) their biological activity is modulated by proprotein convertase-mediated proteolysis; (3) their LRP-6 receptors are required for the proper onset of DA neurogenesis in the VM.

We found that Escherichia coli-produced recombinant full-length Dkk- 1 and Dkk-2 decreased the number of TH+ neurons in rat E14.5 VM precursor cultures. However, partially purified Dkk cm. (Mao et al. , 2001a) produced in mammalian cells distinctively regulated DA differentiation. P.p. Dkk-1 had no effect on DA neurogenesis and p.p. Dkk-2 increased the differentiation of Nurr-1+ precursors into TH+ DA neurons. Our results provide indication, that, upon proteolysis of the full-length Dkk-2 and in the presence of Wnts, the second CRD of Dkk-2 might be responsible for the activating effects of the Dkk-2 on canonical Wnt signalling and DA neurogenesis .

Furin and other proprotein convertases are involved in the proteolytic maturation of several proteins, such as the D-nerve growth factor, Notch, α-amyloid precursor protein and members of the tumor necrosis factor (TNF) and transforming growth factor β (TGF-β) families (Thomas, 2002) . Pro-BMP-4 undergoes sequential cleavage at two furin sites, which renders the mature BMP-4 more stable (not targeted to degradation) , more active and capable of long range signalling (Cui et al . , 2001; Degnin et al . , 2004) . Our experiments with the general proprotein convertase inhibitor decanoyl-RVKR-CMK provide indication that cleavage of full-length Dkks by members of the proprotein convertase family critically regulate their ability to modulate canonical Wnt signalling and, consequently, regulate biological processes including DA neurogenesis. Interestingly, proprotein convertases are highly expressed in the VM during neurogenesis (Zheng et al . , 1994) and our data indicates that the DA neurogenic niche contains high levels of Dkk-1 and Dkk-2.

We hypothesize that Dkk-1 and Dkk-2 inhibit the differentiation of DA precursors at early stages in the VM. At the onset of DA neurogenesis, furin or PC2 mediated cleavage might render Dkk-1 inactive and convert Dkk-2 into an inducer of the canonical Wnt pathway and DA differentiation in the VM, through its second CRD and in the presence of Wnts (Castelo-Branco et al. , 2003) . This hypothesis is consistent with previous reports in Xenopus, where in the absence of Kremen-2 and in the presence of high levels of LRP-6 (Brott and Sokol, 2002; Mao and Niehrs, 2003) , Dkk-2 can activate canonical Wnt signalling. Furthermore, Dkk-2-CRD-2 can synergize with LRP-6 in secondary axis induction in Xenopus and expression of Wnt/D-catenin target genes (Brott and Sokol, 2002) , and activate TCF/LEF-mediated transcription in mammalian cells overexpressing LRP-6 (Li et al., 2002) . The effects of Dkk-2-CRD-2 as an activator are also in agreement with our previous observation that β-catenin stabilization induces DA differentiation (Castelo-Branco et al. , 2004) . Furthermore, it was shown recently that covalently linked chimeras containing Wnt-3a and Dkk-2-CRD-2 activate canonical signalling (Liu et al. , 2005) . Our results also indicate that Wnt-3a and Dkk-2-CRD-2 can indeed synergize to activate canonical Wnt signalling, suggesting that Wnts and truncated Dkks can together modulate diverse signalling pathways and thus regulate DA neurogenesis .

The positive effects of Dkks-CRD-1 in DA neurogenesis were also surprising, since the first CRDs of Dkks are not able to modulate canonical Wnt signalling (Brott and Sokol, 2002; Li et al. , 2002) and can not interact with LRP-6 (Brott and Sokol, 2002; Mao and Niehrs, 2003) . Our results indicate that these proteins modulate DA neurogenesis and could therefore be applied to promote the DA differentiation of neural stem or progenitor cells for therapeutical purposes .

We also analysed DA neurogenesis in the VM of mice mutant for the LRP-6 receptor. Importantly, we found a delayed onset of DA differentiation, with 50% less DA neurons at Ell.5 and a 25% reduction at E13.5. The expression levels of two key transcription factors involved in DA differentiation, Nurr-1 and Pitx-3, were also transiently lower. These results highlight that the elimination of the Dkk receptor LRP-6 in the VM, which mimics excessive Dkk-1 signalling or diminished Dkk-2 and canonical Wnt signalling, perturbs DA differentiation. In contrast, we did not observe any difference in cell death, proliferation or patterning, processes that have been also described to be regulated by Wnts (Castelo- Branco et al. , 2003; Ciani and Salinas, 2005; McMahon and Bradley, 1990; Panhuysen et al . , 2004; Pinson et al . , 2000,- Thomas and Capecchi, 1990) . Similar results showing no difference in cell death and proliferation have been reported in the presomitic mesoderm of LRP-6 hypomorphic mice (Kokubu et al . , 2004) . The apparent selectivity in the functions regulated by Wnts in the LRP-6 homozygote mutant mice has also been observed in other brain areas. Dorsal thalamic development is severely affected in the LRP-6 homozygote mutant (with ablation of Shh and Wnt-5a expression) (Zhou et al . , 2004a) . LRP-6 homozygote mutant mice have also been reported to display neural precursors defects in the dentate gyrus, while other cell types in the developing hippocampus are not affected (Zhou et al . , 2004b) . Thus, our results, showing that lack of LRP-6 affects DA neurogenesis highlight LRP-6 as a general regulator of cell type-specific neurogenesis.

Interestingly, after the initial defect in DA neurogenesis in the LRP6^"/" mice, we found an almost complete recovery of the substantia nigra and ventral tegmental area by E17.5. These results provide indication of the presence of compensatory mechanisms that permit a recovery in the number of DA neurons and expression of DA markers at later stages. One such possible mechanism of compensation may involve LRP5 since the expression of is receptor is up-regulated in the LRP6^"/" mice.

In sum, our results indicate that LRP5/6 regulate the onset of DA neurogenesis. We also found that post-translational processing of LRP6 ligands differentially modulate Wnt/β-catenin signalling. Importantly, we identify proteolytically processed Dkk2, Dkk3 and Dkk-2-CRD-2 as candidates to enhance the differentiation of DA precursors into neurons. These factors might therefore be useful in promoting DA neurogenesis in the context of (stem) cell replacement approaches for the treatment of Parkinson' s disease. References

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Table 1

Claims

CLAIMS :

1. A method of inducing or promoting dopaminergic neuronal development by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and/or maturation in a neural stem, embryonic stem, progenitor or precursor cell, the method comprising: modulating the level and/or activity of a Dkk ligand/receptor polypeptide in said cell, thereby producing or enhancing proliferation, self-renewal, survival and/or dopaminergic induction, differentiation, survival or acquisition of a neuronal dopaminergic phenotype.

2. A method according to claim 1 comprising treating the cell with a Dkk ligand/receptor polypeptide.

3. A method according to claim 2 wherein the Dkk ligand/receptor polypeptide is a Dkkl, Dkk2, Dkk-3 or Dkk4 polypeptide

4. A method according to claim 3 wherein the polypeptide comprises cysteine rich domain 1 and/or cysteine rich domain 2 from human Dkkl, Dkk2, Dkk3 or Dkk4.

5. A method according to claim 3 or claim 4 wherein the Dkk polypeptide is a Dkkl polypeptide having at least 50% sequence identity to human Dkk-1.

6. A method according to claim 3 or claim 4 wherein the Dkk-2 polypeptide has at least 50% sequence identity to human Dkk-2.

7. A method according to claim 3 or claim 4 wherein the Dkk-3 polypeptide has at least 50% sequence identity to human Dkk-3.

8. A method according to claim 3 or claim 4 wherein the Dkk-4 polypeptide has at least 50% sequence identity to human Dkk-4.

9. A method according to claim 1 or claim 2 wherein the Dkk ligand/receptor polypeptide is an LRP polypeptide.

10. A method according to claim 9 wherein the LRP polypeptide is an LRP-5 polypeptide comprising an amino acid sequence having at least 50% sequence identity to the extra-cellular domain of human LRP-5

11. A method according to claim 10 wherein the LRP polypeptide is an LRP-5 polypeptide comprises the extra-cellular domain of human LRP-5

12. A method according to claim 10 or claim 11 wherein the LRP polypeptide comprises an amino acid sequence having at least 50% sequence identity to human LRP-5.

13. A method according to claim 12 wherein the LRP polypeptide comprises the amino acid sequence of human LRP-5.

14. A method according to claim 9 wherein the LRP polypeptide is an LRP-6 polypeptide comprising an amino acid sequence having at least 50% sequence identity to the extra-cellular domain of human LRP-6.

15. A method according to claim 14 wherein the LRP polypeptide is an LRP-6 polypeptide comprises the extra-cellular domain of human LRP-6.

16. A method according to claim 14 or claim 15 wherein the LRP polypeptide comprises an amino acid sequence having at least 50% sequence identity to human LRP-6.

17. A method according to claim 16 wherein the LRP polypeptide comprises the amino acid sequence of human LRP-6.

18. A method according to any one of claims 3 to 17 wherein the Dkk ligand/receptor polypeptide further comprises one or more additional amino acids .

19. A method according to claim 7 wherein the Dkk ligand/receptor polypeptide further comprises an immunoglobulin Fc domain.

20. A method according to any one of claims 2 to 19 wherein the Dkk ligand/receptor polypeptide is added to an in vitro culture containing the cell.

21. A method according to any one of claims 2 to 19 wherein the Dkk ligand/receptor polypeptide is produced by expression from a cell co-cultured with the stem, embryonic stem, neural stem, progenitor or precursor cell, which co-cultured cell is a cell other than a type 1 astrocyte or early glial cell, or is a host cell transformed with nucleic acid encoding the Dkk ligand/receptor polypeptide or a cell containing introduced Dkk ligand/receptor polypeptide.

22. A method according to claim 21 wherein the co-cultured cell other than a type 1 astrocyte or early glial cell or host cell is another stem, embryonic stem, neural stem, progenitor, precursor or neural cell.

23. A method according to claim 22 wherein the stem, embryonic stem, neural stem, progenitor or precursor cell, or other stem or neural cell, is engineered to express the Dkk ligand/receptor polypeptide from encoding nucleic acid.

24 A method according to claim 22 wherein the Dkk ligand/receptor polypeptide is introduced into the co-cultured cell.

25. A method according to claim 1 comprising treating the cell with a non-peptidyl Dkk ligand.

26. A method according to claim 1 comprising treating the cell with a compound which is an inhibitor, blocker, antagonist or suppressor of a Dkk ligand/receptor polypeptide.

27. A method according to claim 26 wherein the compound which is an inhibitor, blocker, antagonist or suppressor of Dkkl or Dkk4.

28. A method according to claim 1 wherein the level of said Dkk ligand/receptor polypeptide is modulated by modulating the activity of a proprotein convertase in said cell.

29. A method according to claim 28 wherein the proprotein convertase specifically cleaves the sequence Arg-X-X-Arg, Arg/Lys-X-X-X-Arg/Lys-Arg, or Arg/Lys-Arg.

30. A method according to claim 29 wherein the proprotein convertase is furin or protease convertase 2 (PC2) .

31. A method according to any one of the preceding claims wherein the cell expresses a nuclear receptor of the Nurrl subfamily.

32. A method according to claim 31 wherein the nuclear receptor is Nurrl .

33. A method according to any one of the preceding claims wherein the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is mitotic and/or capable of self-renewal .

34. A method according to any one of the preceding claims wherein said stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is additionally contacted with a member of the Wnt family of ligands or an upstream regulator of Writs, such as Msxl.

35. A method according to any one of the preceding claims wherein said stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is contacted with a retinoid or retinoid derivative, an activator of the retinoid X receptor (RXR) , a repressor of the retinoid acid receptor (RAR), 9-cis retinal, DHA, SR11237, or LG849

36. A method according to any one of the preceding claims wherein the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is treated with a member of the FGF family of growth factors prior to or simultaneously with modulating the level and/or activity of the Dkk ligand/receptor polypeptide in the cell.

37. A method according to claim 36 wherein the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is treated with bFGF and/or EGF and/or FGF-8 and/or LIF and/or Shh and/or insulin.

38. A method according to any one of the preceding claims wherein the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is grown in the presence of antioxidants, ascorbic acid, low oxygen tension, a hypoxia-induced factor, neurotrophins, GDNF, engrailed, Pax2, Mashl, neurogenins, Pitx3 or BcIxL.

39. A method according to any one of the preceding claims wherein the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell grows and/or differentiates in the presence of ventral mesencephalic astrocytes or early glial cells.

40. A method according to any one of the preceding claims comprising further co-culturing the stem, embryonic stem, neural stem, progenitor or precursor cell, or other stem or neural cell, with an early glial cell, or a Type 1 astrocyte optionally of the ventral mesencephalon.

41. A method according to claim 40 wherein the Type 1 astrocyte is immortalized or is of an astrocyte cell line of a region other than the ventral mesencephalon.

42. A method according to any one of the preceding claims, comprising additionally contacting the stem, embryonic stem, neural stem, progenitor or precursor cell, or other stem or neural cell with a negative selection agent that selects against non-dopaminergic neurons.

43. A method according to any one of the preceding claims comprising subjecting the neural stem, embryonic stem, progenitor or precursor cell to oxidative stress before modulating the level and/or activity of a Dkk ligand/receptor polypeptide in the cell.

44. A method according to any one of the preceding claims further comprising formulating the treated neuron into a composition comprising one or more additional components.

45. A method according to claim 44 wherein the composition comprises a pharmaceutically acceptable excipient.

46. A method according to claim 45 further comprising administering the composition to an individual .

47. A method according to claim 46 wherein the neuron is implanted into the brain of the individual.

48. A method according to any one of claims 2 to 19 wherein the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is treated in an individual in situ with a Dkk ligand/receptor polypeptide.

^•

49. A method according to claim 48 wherein nucleic acid encoding the Dkk ligand/receptor polypeptide is introduced into the cell .

50. A method according to claim 48 wherein Dkk ligand/receptor polypeptide is introduced into the cell.

51. A method according to any one of claims 48 to 50 wherein the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is endogenous to the individual .

52. A method according to any one of claims 48 to 50 wherein the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is exogenously supplied by grafting into the individual.

53. A method according to any one of claims 48 to 52 wherein the individual has Parkinson's disease, a parkinsonian syndrome, neuronal loss or a neurodegenerative disease.

54. A method according to any one of claims 1 to 43 further comprising using the neuron produced in accordance with the method in the manufacture of a medicament for treatment of an individual.

55. A method according to claim 54 wherein the medicament is for implantation into the brain of the individual .

56. A method according to claim 55 wherein the individual has Parkinson's disease, a parkinsonian syndrome, neuronal loss or a neurodegenerative disease.

57. A dopaminergic neuron produced in accordance with any one of claims 1 to 43.

58. Use of a dopaminergic neuron according to claim 57 in a method of screening for an agent for use in treatment of a neurodegenerative disease.

59. A method of screening for an agent for use in treatment of a neurodegenerative disease comprising:

(i) treating a dopaminergic neuron produced by a method according to any one of claims 1 to 43 with a toxin for said dopaminergic neuron;

(ii) separating the dopaminergic neuron from the toxin,- (iϋ) bringing the dopaminergic neuron into contact with a test agent or test agents;

(iv) determining the ability of the dopaminergic neuron to recover from the toxin,-

(v) comparing said ability of the dopaminergic neuron to recover from the toxin with the ability of a dopaminergic neuron to recover from the toxin in the absence of contact with the test agent or test agents.

60. A method of screening for an agent for use in treatment of a neurodegenerative disease comprising:

(i) treating a dopaminergic neuron according to claim 57 with a toxin for the dopaminergic neuron in the presence of a test agent or test agents;

(ii) determining the ability of the dopaminergic neuron to tolerate the toxin;

(iii) comparing said ability of the dopaminergic neuron to tolerate the toxin with the ability of a dopaminergic neuron to tolerate the toxin in the absence of contact with the test agent or test agents .

61. A method of screening for an agent for use in the treatment of a neurodegenerative disease comprising; (i) bringing a dopaminergic neuron produced by a method according to any one of claims 1 to 43 into contact with a test agent or test agents;

(ii) determining the growth or viability or neurotransmitter release of the dopaminergic neuron in the presence of said agent or agents.

62. A method according to claim 61 wherein the dopaminergic neuron is generated from a stem, progenitor or precursor cell subjected to oxidative stress.

63. A method according to any one of claims 60 to 62 further comprising formulating the test agent or test agents into a composition comprising one or more additional components.

64. A method according to claim 63 wherein said composition comprises a pharmaceutically acceptable excipient.

65. A method according to claim 64 further comprising administering said composition to an individual.

66. A method according to claim 65 wherein the individual has Parkinson's disease, a parkinsonian syndrome, neuronal loss or a neurodegenerative disease.

67. A method of obtaining a factor or factors which, either alone or in combination, enhance proliferation, self-renewal, survival and/or dopaminergic neurotransmission, development, induction, differentiation, or maturation in a stem, embryonic stem, neural stem, progenitor or precursor cell, the method comprising:

(a) treating a neural stem progenitor or precursor cell, with a Dkk ligand or Dkk receptor in the presence and absence of one or more test substances; and (b) determining proliferation, self-renewal, survival and/or dopaminergic neurotransmission, development, induction, differentiation, or maturation of the cell and comparing the extent of the proliferation, self-renewal, survival and/or dopaminergic neurotransmission, development, induction, differentiation or maturation in the presence and absence of the test substance or substances, whereby said factor or factors is obtained.

68. A method of obtaining a factor or factors which, either alone or in combination, enhance proliferation, self-renewal, survival and/or dopaminergic neurotransmission, development, induction, differentiation, or maturation in a stem, embryonic stem, neural stem, progenitor or precursor cell, the method comprising:

(b) treating a neural stem progenitor or precursor cell, with a Dkk ligand or Dkk receptor in the presence and absence of one or more test substances; and (b) determining β-catenin activation in the cell and comparing the extent of said β-catenin activation activation in the presence and absence of the test substance or substances, whereby said factor or factors is obtained.

69. A method according to claim 67 or claim 68 wherein the neural stem progenitor or precursor cell is treated with a Dkk ligand/receptor polypeptide.

70. A method according to claim 69 wherein the Dkk ligand/receptor polypeptide is a Dkkl, Dkk-2, Dkk3 or Dkk4 polypeptide

71. A method according to claim 69 or claim 70 wherein Dkk ligand/receptor polypeptide comprises cysteine rich domain 1 and/or cysteine rich domain 2 from human Dkkl, Dkk2, Dkk3 or Dkk4.

72. A method according to claim 70 or 71 wherein the Dkk ligand/receptor polypeptide is a Dkkl polypeptide having at least 50% sequence identity to human Dkk-1.

73. A method according to claim 70 or 71 wherein the Dkk ligand/receptor polypeptide is a Dkk-2 polypeptide having at least 50% sequence identity to human Dkk-2.

74. A method according to claim 70 or 71 wherein the Dkk ligand/receptor polypeptide is a Dkk-3 polypeptide having at least 50% sequence identity to human Dkk-3.

75. A method according to claim 70 or 71 wherein the Dkk ligand/receptor polypeptide is a Dkk-4 polypeptide having at least 50% sequence identity to human Dkk-4.

76. A method according to claim 69 wherein the Dkk ligand/receptor polypeptide is an LRP polypeptide.

77. A method according to claim 76 wherein the LRP polypeptide is an LRP-5 polypeptide comprising an amino acid sequence having at least 50% sequence identity to the extracellular domain of human LRP-5

78. A method according to claim 77 wherein the LRP polypeptide is an LRP-5 polypeptide comprising the extracellular domain of human LRP-5

79. A method according to claim 77 or claim 78 wherein the LRP polypeptide comprises an amino acid sequence having at least 50% sequence identity to human LRP-5.

80. A method according to claim 79 wherein the LRP polypeptide comprises the amino acid sequence of human LRP-5.

81. A method according to claim 76 wherein the LRP polypeptide is an LRP-6 polypeptide comprising an amino acid sequence having at least 50% sequence identity to the extracellular domain of human LRP-6.

82. A method according to claim 81 wherein the LRP polypeptide is an LRP-6 polypeptide comprises the extracellular domain of human LRP-6.

83. A method according to claim 81 or claim 82 wherein the LRP polypeptide comprises an amino acid sequence having at least 50% sequence identity to human LRP-6.

84. A method according to claim 83 wherein the LRP polypeptide comprises the amino acid sequence of human LRP-6.

85. A method according to any one of claims 76 to 84 wherein the LRP polypeptide further comprises one or more additional amino acids.

86. A method according to claim 85 wherein the LRP polypeptide further comprises an immunoglobulin Fc domain.

87. A method according to any one of claims 68 to 86 wherein the cell is treated with the Dkk ligand/receptor polypeptide by addition of the Dkk ligand/receptor polypeptide to in vitro culture containing the cell.

88. A method according to any one of claims 68 to 86 wherein the cell is treated with the Dkk ligand/receptor polypeptide by introduction of nucleic acid encoding the Dkk ligand/receptor polypeptide into the cell .

89. A method according to any one of claims 68 to 86 wherein the cell is treated with the Dkk ligand/receptor polypeptide by introduction of Dkk ligand/receptor polypeptide into the cell.

90. A method according to any one of claims 68 to 86 wherein the stem, embryonic stem, neural stem, progenitor or precursor cell, or other stem or neural cell is treated with the Dkk ligand/receptor polypeptide by co-culturing with a cell which is a cell other than a type 1 astrocyte or early glial cell or is a host cell transformed with nucleic acid encoding the Dkk ligand/receptor polypeptide or a cell containing introduced Dkk ligand/receptor polypeptide.

91. A method according to any one of claims 68 to 86 further comprising co-culturing the stem, embryonic stem, neural stem, progenitor or precursor cell, or other stem or neural cell with an early glial cell or a Type 1 astrocyte optionally of the ventral mesencephalon.

92. A method of screening for a compound useful in inducing or promoting dopaminergic neuronal development by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and/or maturation in a neural stem, embryonic stem, progenitor or precursor cell, the method comprising; determining the ability of a proprotein convertase to proteolytically cleave a Dkk polypeptide in the presence of a test compound, wherein an increase or decrease in said ability in the presence relative to the absence of the test compound is indicative that the test compound is useful in inducing or promoting dopaminergic neuronal development.

93. A method according to claim 92 wherein the ability of a proprotein convertase to proteolytically cleave a Dkk polypeptide is determined by determining Dkk activity in the presence of the proprotein convertase.

94. A method according to claim 93 wherein Dkk activity is determined by determining β-catenin activation or doperminergic development.

95. A method according to claim 92 wherein the ability of a proprotein convertase to proteolytically cleave a Dkk polypeptide is determined by determining the presence of products of proteolytic cleavage of the Dkk polypeptide.

96. A method according to any one of claims 92 to 95 wherein the Dkk polypeptide is Dkk-1, Dkk-2, Dkk-3 and/or Dkk-4.

97. A method according to any one of claims 92 to 96 wherein the proprotein convertase is specific for the sequence Arg-X- X-Arg, Arg/Lys-X-X-X-Arg/Lys-Arg, or Arg/Lys-Arg.

98. A method according to claim 97 wherein the proprotein convertase is furin or protease convertase 2 (PC2) .

99. A method according to any one of claims 68 to 98 wherein a compound, factor or factors able to enhance proliferation, self-renewal, survival and/or dopaminergic neurotransmission, development, induction, differentiation or maturation in a stem, embryonic stem, neural stem, progenitor or precursor cell is or are provided in isolated and/or purified form.

100. A method according to any one of claims 68 to 99 wherein a compound, factor or factors able to enhance proliferation, self-renewal, survival and/or dopaminergic neurotransmission, development, induction, differentiation or maturation in a stem, embryonic stem, neural stem, progenitor or precursor cell is or are formulated into a composition comprising one or more additional components.

101. A method according to claim 102 wherein the composition comprises a stem, embryonic stem, neural stem, progenitor or precursor cell.

102. A method according to claim 100 or 101 wherein the composition comprises a Dkk ligand/receptor polypeptide.

103. A method according to any one of claims 100 to 102 wherein the composition comprises a pharmaceutically acceptable excipient.

104. A method according to claim 103 further comprising administering the composition to an individual.

105. A method according to claim 104 wherein the composition is implanted into the brain of the individual .

106. A method according to claim 105 wherein the individual has Parkinson's disease, a parkinsonian syndrome, neuronal loss or a neurodegenerative disease.

107. A method of screening for a Dkk ligand which is not susceptible to proteolysis comprising; determining the Dkk activity of sample suspected of containing the Dkk ligand in the presence of a proprotein convertase, the presence of activity being indicative that the sample contains said Dkk ligand.

108. A method according to claim 107 wherein the Dkk activity of the sample is determined by determining the effect of said sample on promoting dopaminergic neuronal development by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and/or maturation in a neural stem, embryonic stem, progenitor or precursor cell,

109. A method according to claim 107 wherein the Dkk activity of the sample is determined by determining β-catenin activation.

110. A method according to claim 107 wherein the Dkk activity of the sample is determined by determining the presence of products of proteolytic cleavage of the Dkk ligand.

111. A method according to any one of claims 107 to 110 wherein the proprotein convertase is specific for the sequence Arg-X-X-Arg, Arg/Lys-X-X-X-Arg/Lys-Arg, or Arg/Lys-Arg.

112. A method according to claim 111 wherein the proprotein convertase is furin or protease convertase 2 (PC2) .

113. A method according to any one of claims 107 to 112 comprising identifying a sample as containing a Dkk ligand which is not susceptible to proteolysis.

114. A method according to claim 113 comprising isolating and/or purifying the Dkk ligand from the sample.