JP2008522607A - Dickkopfs and materials and methods related to neurogenesis - Google Patents

Abstract

  A method for producing neurons that promote the development of dopaminergic neurons and have a dopaminergic phenotype. Dopaminergic neurons can be used to treat individuals with neurodegenerative diseases such as Parkinson's disease. Dopaminergic cells can be transplanted into the individual's brain and / or the development of dopaminergic nerves can be induced or enhanced in the individual's brain. These methods treat cells with a Dkk ligand (eg, any one of Dkk1 to Dkk4, or a fragment thereof containing a cysteine-rich domain) or a Dkk receptor (eg, LRP5 or LRP6), thereby Inducing or enhancing proliferation, self-renewal, survival and / or induction of dopaminergic, differentiation, survival or acquisition of a neuron's dopaminergic phenotype. Cells may be co-cultured with astrocytes or glial cells or contacted with FGF growth factors. Dopaminergic neurons are useful for drug / toxicology testing and may also be useful for target development and drug discovery.

Description

  The present invention relates to inducing the fate of neurons (neural cells) in neural stem cells or neural progenitor or progenitor cells, or other stem cells. The present invention relates to inducing and enhancing the induction of specific neuronal phenotypes, and in particular to inducing and enhancing the phenotype of midbrain dopaminergic neurons.

  Parkinson's disease (PD) is a very common neurodegenerative disorder characterized by selective and progressive reduction of midbrain dopaminergic (DA) neurons. Increasing the induction of the neuronal phenotype has the potential to treat Parkinson's disease and other highly debilitating neurodegenerative disorders.

  Previously, human fetal mesencephalic tissue was transplanted into Parkinson's disease patients with positive results, but the development of specific cell replacement therapies utilizing the present invention has led to the actuality associated with such previous approaches. Top and ethical issues are overcome. In particular, the present invention allows the development of cell preparations for transplantation while reducing or eliminating the need to use embryonic tissue or fetal cells. Stem cells can be obtained from the umbilical cord, which is usually discarded tissue. Another option is to obtain adult stem cells, which are obtained from, for example, bone marrow, blood, skin, eyes, olfactory bulb or olfactory epithelium.

  Induction of specific neuronal phenotypes requires the integration of both genetic and epigenetic signals. In the developing midbrain, it has been reported that the orphan nuclear receptor Nurr1 is required for induction of dopaminergic neurons (Zetterstroem et al., 1997; Saucedo-Cardenas et al., 1998; Castillo et al., 1998). However, Nurr1 expression is not sufficient to induce a dopaminergic phenotype in neural stem cells (Wagner et al., 1999).

  The findings described herein indicate that Dickkopf (Dkk) ligands and receptors are critical regulators of proliferation, self-renewal, differentiation and fate decisions in ventral midbrain neurogenesis. In addition, our results pave the way for large-scale production of midbrain DA neurons in vitro and the future realization of stem cell replacement strategies in treating 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 stem cells, neural stem cells, and pluripotent stem cells are capable of differentiating into neural cell lineages, including neurons, astrocytes, and oligodendrocytes. In addition, stem cells can be isolated and expanded and then used as a source material for transplantation into the brain (Snyder, EY et al. Cell 68, 33-51 (1992); Rosenthal, A. Neuron 20, 169- 172 (1998); Bain et al., 1995; Gage, FH et al. Ann. Rev. Neurosci. 18, 159-192 (1995); Okabe et al., 1996; Weiss, S. et al. Trends Neurosci. 19, 387-393 (1996) Snyder, EY 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 the neuronal population. In addition, most of the damage occurs in cells with a specific neurochemical phenotype. For example, in human Parkinson's disease, the predominantly lost cell type 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); Bjoerklund A. et al., Lancet Neurol. 2 (7) : 437-45 (2003)). Another alternative is to directly inject signals necessary to promote regeneration, repair, or to induce the development and / or recruitment of stem, progenitor or progenitor cells, or It would be to administer drugs that modulate these functions.

  Stem / progenitor or progenitor cells are ideal materials for transplantation therapy because they can be augmented to command a specific neuronal phenotype. These cells will avoid ethical and practical problems associated with using human fetal tissue for transplantation.

  It has been found that inducing a single specific neuronal phenotype in stem cells or progenitor or progenitor cells is not relevant.

  The present invention provides for the induction of dopaminergic neuronal phenotypes in cells. Thus, Dickkopf ligands (eg, Dickkopf-1, -2, -3 or -4 (Dkks)) and / or Dkk receptors (eg, low density lipoprotein receptor (LDLR) related protein-5 in culture or brain) Or -6 (LRP5 / 6)), the present invention allows the induction and promotion of dopaminergic progenitor, progenitor or stem cell proliferation and / or self-renewal; and / or dopaminergic Promote survival, differentiation and maturation of neurons, progenitor cells, progenitor cells or stem cells; increase production of dopaminergic neurons; and / or induce dopaminergic neuron fate in stem cells, progenitor cells, progenitor cells or neuronal cells Allows in vitro or in vivo.

  Any aspect or embodiment of the present invention can be applied to, or can use, stem cells, progenitor cells, progenitor cells or neurons or neurons. As used herein, “neural cells” may be neuronal cells.

  Cell preparations rich in dopaminergic neurons can be used for cell replacement therapy in Parkinson's disease and other disorders, as well as signal transduction events in dopaminergic neurons and the effects of drugs on dopaminergic neurons. It can be used to study in vitro (eg in high throughput screening).

  Aspects and embodiments of the invention are provided as set forth in the claims.

In one aspect, the invention provides a method of inducing dopaminergic neuron fate in a stem cell, neural stem cell, embryonic stem cell, or neural progenitor or progenitor cell, or a method of enhancing dopaminergic induction or differentiation in neuronal cells, Or providing a method of increasing dopaminergic progenitor or progenitor cells, the method comprising:
Modulate the level and / or activity of a Dkk ligand / receptor polypeptide in the cell;
Thereby producing dopaminergic neurons,
Comprising that.

The present invention induces the generation of dopaminergic neurons by enhancing proliferation, self-replication, dopaminergic induction, survival, differentiation and / or maturation in neural stem cells, progenitor or progenitor cells, or other stem cells or neural cells Or provide a way to promote,
Modulate the level and / or activity of a Dkk ligand / receptor polypeptide in the cell;
Thereby causing or enhancing proliferation, self-renewal, survival, and / or induction of dopaminergic, differentiation, neurotransmission, survival or acquisition of a neuron's dopaminergic phenotype,
Comprising that.

  A Dkk ligand / receptor polypeptide is a polypeptide that is capable of forming a Dkk receptor / ligand complex with an appropriate binding partner. Dkk ligand / receptor polypeptides include polypeptides that are Dkk ligands and polypeptides that are Dkk receptors. Examples of Dkk ligand / receptor polypeptides include Dkk polypeptides that bind to Dkk receptors, such as Dkk-1, Dkk-2, Dkk-3 or Dkk-4, and LRP5, LRP6 that bind to Dkk polypeptides. , Dkk receptors such as Kremen 1 or Kremen 2. The Dkk ligand / receptor polypeptide may be an endogenous cellular polypeptide or a synthetic polypeptide made recombinantly or by other means.

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

  Dkk ligands are peptidic or non-peptidic molecules (ie, Dkk receptor agonists) that can form Dkk receptor / ligand complexes with Dkk receptors. Examples of Dkk ligands include Dkk polypeptides as described below.

  The Dkk polypeptide may be a Dkk-1, -2, -3 or -4 polypeptide from a mammalian species, such as mouse Dkk-1, -2, -3 or -4, or human Dkk-1,- 2, -3, or -4, and the Dkk polypeptide may be any fragment or variant thereof. For example, a Dkk-1 polypeptide can include a proteolytic fragment as shown in FIG. 11, and a Dkk-2 polypeptide can include a proteolytic fragment as shown in FIG.

  A Dkk polypeptide may comprise one or both of two cysteine rich domains (Cys-1 or Cys-2) of a wild type Dkk sequence such as human Dkk1, Dkk2, Dkk3 or Dkk4. It may also consist of, or may comprise one or both variants of these domains or consist of such variants.

  The Cys-1 domain of human Dkk1 consists of residues 85-138 of the full-length sequence, and the Cys-2 domain of human Dkk1 consists of residues 189-263 of the full-length sequence.

  The Cys-1 domain of human Dkk2 consists of residues 78-127 of the full length sequence, and the Cys-2 domain of human Dkk2 consists of residues 183-156 of the full length sequence.

  The Cys-1 domain of human Dkk3 consists of residues 147-195 of the full-length sequence, and the Cys-2 domain of human Dkk3 consists of residues 208-284 of the full-length sequence.

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

  Fragments or variants of the wild-type Dkk sequences described herein retain the ability to modulate the development of dopaminergic neuron fate in stem cells, neural stem cells, embryonic stem cells, or neural progenitor or progenitor cells May differ from the wild-type sequence by addition, deletion, substitution and / or insertion of one or more amino acids (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 that is a variant of a wild type sequence has a wild type sequence (eg, human Dkk-1, -2, -3, or -4) or about 30% or more of about one or more domains, about Containing amino acid sequences having sequence identity of 40% or more, about 45% or more, about 55% or more, about 65% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more It can be. The sequence is about 30% or more, about 40% or more, about 50% or more, about 60 for a wild type sequence (eg, human Dkk-1, -2, -3, or -4) or one or more domains thereof. % Or more, about 70% or more, about 80% or more, or about 90% or more similarity.

  The amino acid sequence of human Dkk-1 can be obtained from GenBank reference Swiss protein accession number AAF02674.1 (GI: 6049604) and the nucleic acid encoded by it can be obtained from reference AF177394.1 (GI: 6049603).

  The amino acid sequence of human Dkk-2 is available from GenBank reference Swiss protein accession number AAQ88780.1 (GI: 37181953) and the nucleic acid encoded by it is available from reference AY358414.1 (GI: 37181952).

  The amino acid sequence of human Dkk-3 can be obtained from GenBank reference Swiss protein accession number AAF02676.1 (GI: 6049608), and the nucleic acid encoded by it can be obtained from reference AF177396.1 (GI: 6049607).

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

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

  The LRP polypeptide may include an LRP sequence derived from a mammalian species (eg, mouse LRP or human LRP), or may include a sequence that is a fragment or variant thereof.

  A Kremen polypeptide may include a Kremen sequence (eg, mouse Kremen or human Kremen) from a mammalian species, or a sequence that is a fragment or variant thereof.

  One fragment or variant of wild-type LRP or Kremen sequence, provided that the ability to enhance the fate development of dopaminergic neurons is retained in stem cells, neural stem cells, embryonic stem cells, or neural progenitor or progenitor cells Or may differ from the wild-type sequence by the addition, deletion, substitution and / or insertion of multiple amino acids (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, the LRP polypeptide is about 30% or more, about 40% or more, about 45% or more, about 55% or more, about 65% or more, relative to the extracellular domain or full-length sequence of human LRP-5 or LRP-6, It may comprise amino acid sequences having about 70% or more, about 80% or more, about 90% or more, or about 95% or more sequence identity. This sequence is about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more with respect to the extracellular domain or full-length sequence of human LRP-5 or LRP-6 Or a similarity of about 90% or more.

  The amino acid sequence of human LRP-5 is available from GenBank reference accession number NP — 002326.1 (GI: 4505019), and the encoding nucleic acid is available from the reference NM — 002335.1 (GI: 4505018) for DNA. The extracellular domain of Homo sapiens LRP-5 is encoded by a 4100 base pair region starting with the first ATG.

  The amino acid sequence of human LRP-6 is available from GenBank reference accession number NP — 002327.1 (GI: 4505017), and the encoding nucleic acid is available from the reference NM — 002336.1 (GI: 4505016) for DNA. The extracellular domain of Homo sapiens LRP-6 is encoded by a 4167 base pair region starting with the first ATG.

  The Kremen polypeptide is about 30% or more, about 40% or more, about 45% or more, about 55% or more, about 65% or more, about 70% to the extracellular domain or full-length sequence of human Kremen-1 or Kremen-2. % Or more, about 80% or more, about 90% or more, or about 95% or more of amino acid sequences having sequence identity. This sequence is about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more with respect to the extracellular domain or full-length sequence of human Kremen-1 or Kremen-2 Or a similarity of about 90% or more.

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

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

  In certain preferred embodiments, an LRP or Kremen polypeptide comprises an amino acid sequence that is an extracellular domain of human LRP or Kremen, or a fragment or variant of a human LRP or Kremen extracellular domain, such an amino acid sequence. It may be composed of

  In certain embodiments, the LRP or Kremen polypeptide can be a soluble non-membrane bound polypeptide. This polypeptide lacks the transmembrane and / or intracellular domain of, for example, wild type LRP or Kremen.

  In other embodiments, the LRP or Kremen polypeptide may be membrane bound. This polypeptide includes, for example, the transmembrane and / or intracellular domain of wild type LRP or Kremen.

  The Dkk ligand / receptor polypeptides described herein can further comprise one or more additional amino acids. For example, an LRP or Kremen polypeptide is a fusion comprising an LRP or Kremen amino acid sequence (eg, the extracellular domain of human LRP or Kremen or a fragment or variant thereof) and a peptide component that can confer one or more additional properties. It may be. For example, the fusion includes an immunoglobulin Fc domain.

  Sequence similarity and identity are generally defined with respect to the algorithm GAP (Genetics Computer Group, Madison, Wis.). GAP uses the Needleman and Wunsch algorithm to align two complete sequences to maximize the number of matches and minimize the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and a gap extension penalty = 4. Although the use of GAP is preferred, other algorithms may be used. For example, BLAST (using the method described in Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method described in Pearson and Lipman (1988) PNAS USA 85: 2444-2448) Or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197) or the TBLASTN program (Altschul et al. (1990) supra; generally using default parameters). In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) can be used. Sequence identity and similarity may also be determined using Genomequest ™ software (Gene-IT, Worcester MA USA).

  Preferably, the sequence comparison is performed over the entire length of the related sequence described herein.

  Similarity allows for “conservative mutation”, ie, replacing one hydrophobic residue (eg, isoleucine, valine, leucine, or methionine) with another, or replacing one polar residue with another ( For example, arginine can be replaced with lysine, glutamic acid with aspartic acid, and glutamine with asparagine).

  In certain embodiments, the level and / or activity of a Dkk ligand / receptor polypeptide in a cell can be increased by treating the cell with a Dkk ligand or Dkk receptor. For example, cells are treated with Dkk ligand / receptor polypeptide.

  Cells are treated with Dkk ligand or Dkk receptor in vivo, ex vivo, or in culture (in vitro).

  In the methods described herein, treatment with a Dkk ligand or receptor can be performed by contacting a cell with one or both polypeptides. Treatment with a Dkk ligand or receptor can be performed on a culture containing stem cells, progenitor cells or progenitor cells, or on such cells in vivo, purified and / or recombinant Dkk-2 or -3 polypeptide or other This can be done by supplying a Dkk receptor agonist and / or LRP-5 / 6 receptor or polypeptide. Treatment with a Dkk ligand or receptor can include introducing one or more copies of the encoding nucleic acid or protein into a cell by protein transduction. Methods for transforming cells with nucleic acids and introducing proteins into cells are further described below. Contact with the Dkk ligand or receptor can be made in vivo or by supplying cells producing said polypeptide to a culture comprising stem cells, progenitor or precursor cells, or neuronal cells. A cell that produces a Dkk ligand or receptor can be a recombinant host cell that produces the polypeptide by recombinant expression. A co-cultured host cell may be transformed with a nucleic acid encoding a Dkk ligand or receptor and / or may contain a Dkk ligand or receptor into which the co-cultured host cell has been introduced. Good. Dkk ligands or receptor proteins, or encoding nucleic acids can be introduced into cells according to techniques available in the art, examples of which are described below.

  The co-cultured host cell can be another stem cell, neural stem cell, progenitor cell, progenitor cell, or neural cell. Treatment with a Dkk ligand or receptor may be performed by upregulating the expression of the Dkk ligand or receptor in the cell, or by downregulating or suppressing a Dkk ligand or receptor inhibitor molecule. .

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

  The level and / or activity of a Dkk ligand / receptor polypeptide, such as Dkk-1 or Dkk-4, can also be inhibited by inhibiting the expression of the gene encoding the polypeptide or blocking the polypeptide It can also be reduced by up-regulating or activating agents, antagonists or suppressors.

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

  Suitable suppressors include antisense and sense nucleic acids (eg, 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 PD 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 can also be determined using standard recombinant techniques in embryonic stem cells, neurons, progenitor cells or progenitor cells. It can also be reduced by “knocking out” related genes.

  In other embodiments, the level and / or activity of a Dkk ligand / receptor polypeptide can be modulated by modulating the activity of a proprotein convertase in the cell.

  Examples of precursor protein convertases include proteases that specifically cleave the sequence Arg-XX-Arg, Arg / Lys-XXX-Arg / Lys-Arg, or Arg / Lys-Arg, such as furin or protease convertase 2 ( PC2) is included.

  The activity of the precursor protein convertase can be reduced by treating the cell with an inhibitor or antagonist, and can be increased by treating the cell with an agonist or cofactor.

  A “stem cell” is a cell type that is capable of self-replication and, if it is an embryonic stem (ES) cell, can produce any cell in an individual and is pluripotent Or in the case of neural stem cells, any cell of the nervous system can be generated, including neurons, astrocytes, and oligodendrocytes. Stem cells can express one or more of the following markers: Oct-4; Nanog; Sox1-3; Timing specific fetal antigens (SSEA-1, -3 and -4), and tumor rejection antigen TRA- 1-60 and -1-81 (described in Tropepe et al., 2001; Xu et al., 2001). Neural stem cells can express one or more of the following markers: Nestin; p75 neurotrophin receptor; Notch1, SSEA-1; Sox2; NCAM; RC2 (Capela and Temple, 2002).

  A “neural progenitor cell” is a daughter cell or progeny cell of a neural stem cell and has a more differentiated phenotype and / or a reduced differentiation ability compared to the stem cell. Progenitor cells are other cells that have a direct relationship with neurons in development but can be induced to differentiate or redifferentiate under certain environmental conditions or to acquire a neuronal phenotype . In preferred embodiments, the stem cell, neural stem cell, progenitor cell, progenitor cell, or neuronal cell does not express tyrosine hydroxylase either naturally or upon deficiency of a mitogenic agent (eg, bFGF, EGF or serum), Or it is expressed at a low level.

  Stem cells, neural stem cells, or neural progenitor or progenitor cells can be obtained or derived from any embryonic, fetal or adult tissue, such as bone marrow, skin, eye, nasal epithelium, or umbilical cord, Or a region of the nervous system (eg, from the cerebellum, ventricular zone, subventricular zone, striatum, midbrain, rhinocephalus, cerebral cortex, or hippocampus) is included. It is a vertebrate, such as a mammal (human or non-human animal such as a rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cow, horse, primate), avian (Such as chickens) or amphibians.

  In a preferred embodiment of the present invention, adult stem / progenitor / progenitor cells are used in vitro, ex vivo or in vivo. This requires an consenting adult (obtain cells from that adult) and approval by an appropriate ethics committee. When human embryos / fetuses are used as the source, the human embryos are otherwise discarded or stored indefinitely, especially for couples with IVF (in vitro) that are difficult to conceive. Fertilization) A human embryo created for therapeutic purposes. In general, IVF requires the creation of more human embryos than are used for transplantation and final pregnancy. Such spare embryos are generally discarded. Embryos that would otherwise be abandoned with the consent of interested parties (especially related egg donors and / or sperm donors) in an ethically positive manner for severe neurodegenerative disorders such as Parkinson's disease Can be used for patient benefit. The invention itself is not related to the use of any developmental stage human embryo. As described, the present invention allows the development of valuable therapies for critical diseases while minimizing the need to utilize materials derived directly from human embryos. Any therapeutic intervention based on the present invention should be carried out in accordance with the relevant national laws and ethical guidelines.

  In certain preferred embodiments, stem cells or progenitor or progenitor cells that are contacted with a Dkk ligand or Dkk receptor, or otherwise treated and / or used in accordance with any aspect of the present invention, are consenting adults or appropriate For example, Dkk ligands or Dkk receptors (in order to promote or induce the development or function of neurons generated according to the present invention and / or endogenous dopaminergic neurons ( For example, from a patient having a disease that is substantially treated by back-transplanting neurons treated with a Dkk polypeptide or LRP polypeptide as described above.

  A stem cell or primordial or progenitor cell that seeks to induce neuronal fate may exhibit an undifferentiated phenotype or a primitive neuronal phenotype. It is a totipotent cell that can produce any cell type in an individual, or a pluripotent cell that can produce multiple distinct neuronal phenotypes, or a more limited phenotype during normal development Can be progenitor or progenitor cells that are capable of generating other cells when exposed to appropriate environmental factors in vitro. It may lack markers associated with specific neuronal fate, such as tyrosine hydroxylase.

  The cells can be Nurr1 positive (ie, expressing Nurr1) or Nurr1 negative (ie, not expressing Nurr1). In certain embodiments, cells that are Nurr1 negative will naturally express Nurr1 during differentiation.

  In certain embodiments, the stem cell or progenitor or progenitor cell that is to induce neuronal fate is under oxidative stress. Neurons arising from such cells can be useful as disease cell models. Oxidative stress can be applied to stem cells or progenitor or progenitor cells using conventional toxicological or genetic means.

  In methods that elicit neuronal fate by treating cells with Dkk ligands or Dkk receptors, a large number of cells can be induced to adopt neuronal fate. In neuronal cells, dopaminergic induction or differentiation can be enhanced. In preferred embodiments, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the stem and / or progenitor cells induce neuronal fate Is done.

  Dkk ligand / receptor polypeptides can be expressed from the encoding nucleic acid in situ in stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells, or in vitro in an expression system prior to isolation and purification . The transformed Dkk and / or LRP nucleic acid may be inserted into a vector outside the genome, or preferably stably integrated into the genome. Such nucleic acids can be operably linked to promoters that drive their expression above basal levels in stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells, as described in detail below.

  “Operably linked” means bound, properly positioned and oriented as part of the same nucleic acid molecule such that transcription is initiated from a promoter.

  Methods for introducing genes into cells are well known to those skilled in the art. Nucleic acids encoding Dkk and / or LRP can be introduced into stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells using the vector, whether it remains intact on the vector or integrated into the genome. Appropriate vectors are selected or constructed to contain appropriate regulatory sequences such as promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences. The vector may optionally contain a marker gene and other sequences. Regulatory sequences can drive the expression of nucleic acids encoding Dkk and / or LRP in stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells. For example, the vector may be an extragenomic expression vector, or regulatory sequences may be incorporated into the genome along with nucleic acids encoding Dkk and / or LRP. The vector can be a plasmid or a virus.

  The nucleic acid encoding the Dkk ligand / receptor polypeptide can be placed under the control of an externally inducible gene promoter and placed under user control. 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 type of stimulation varies from promoter to promoter. Some inducible promoters cause little or no detectable level of expression in the absence of appropriate stimuli (ie no expression). Whatever the level of expression in the absence of a stimulus, expression from an inducible promoter will increase in the presence of the appropriate stimulus. An example of an inducible promoter is the tetracycline ON / OFF system (Gossen et al., 1995), where gene expression is regulated by a tetracycline analog.

  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 acids (eg, generation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, protein analysis) are described in Current Protocols in Molecular Biology, Ausubel et al. Ed., John Wiley & Sons, 1992 or later.

  Marker genes such as antibiotic resistance, susceptibility genes, or fluorescent reporters are used to identify clones containing the nucleic acid of interest, which are well known in the art. Clones can also be identified and further studied by binding experiments, Southern blot hybridization, immunohistochemical methods, PCR, and other techniques for detecting protein or nucleic acid expression in cells. it can.

  Nucleic acids encoding Dkk receptor / ligand polypeptides, such as Dkk or LRP polypeptides, can be integrated into the host stem cells, neural stem cells, progenitor or progenitor cells, or the genome of a neuronal cell. Inclusion is facilitated by including sequences in the transformed nucleic acid according to standard techniques that facilitate recombination with the genome. The incorporated nucleic acid can include regulatory sequences capable of driving the expression of nucleic acids encoding Dkk and / or LRP in stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells. The nucleic acid may include a sequence that directs integration into a site in the genome, where the Dkk and / or LRP coding sequence is expressed in stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells. A site that falls under the control of regulatory elements that can drive and / or control expression. The incorporated nucleic acids can be derived from vectors used to transform Dkk and / or LRP nucleic acids into stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells, as described herein.

  Introduction of a nucleic acid comprising a Dkk and / or LRP coding sequence is generally referred to as “transformation” without limitation, whether the nucleic acid is linear, branched, or circular. Any available technique can be used. Suitable techniques include calcium phosphate transfection, mechanical techniques such as DEAE-dextran, PEI, electroporation, microinjection, direct DNA uptake, receptor-mediated DNA transfer, transduction using retroviruses and other viruses, Examples include transfection mediated by liposome-, lipid- or other cationic carriers. When introducing a given gene construct into a cell, certain considerations well known to those skilled in the art must be taken into account. As will be apparent to those skilled in the art, transformation methods for introducing Dkk receptor / ligand polypeptides, such as Dkk and / or LRP polypeptides, into stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells. The particular choice is not essential to the invention and does not limit the invention.

  Suitable vectors and methods for in vitro transformation of stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells with Dkk and / or LRP nucleic acids are well known to those skilled in the art. Suitable vectors include adenovirus, adeno-associated virus, papova virus, vaccinia virus, herpes virus, lentivirus, retrovirus and the like. The disabled virus vector can be produced in a helper cell line in which a gene required for the production of infectious virus particles is expressed. Suitable helper cell lines are well known to those skilled in the art. For example, Fallaux, FJ et al. (1996) Hum Gene Ther 7 (2), 215-222; Willenbrink, W. et al. (1994) J Virol 68 (12), 8413-8417; Cosset, FL et al. (1993) ) Virology 193 (1), 385-395; Highkin, MK et al. (1991) Poult Sci 70 (4), 970-981; Dougherty, JP 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) Mol Cell Biol 4 (9), 1730-1737; Wang, S. et al. (1997) ) Gene Ther 4 (11), 1132-1141; Moore, KW et al. (1990) Science 248 (4960), 1230-1234; Reiss, CS et al. (1987) J Immunol 139 (3), 711-714 I want to be. Helper cell lines generally have lost the sequence recognized by the mechanism that packages the viral genome. They produce virions that are completely free of nucleic acids. Viral vectors containing complete packaging signals along with the gene or sequence to be delivered (eg, Dkk and / or LRP coding sequences) are packaged into infectious virion particles in helper cells, after which these particles are It can be used for gene delivery to stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells.

  As an alternative or in addition to enhancing transcription and / or translation of endogenous Dkk and / or LRP, expression of Dkk and / or LRP beyond basal levels may result in stem cells, neural stem cells, progenitors or progenitors It can also be generated by introducing one or more extra copies of Dkk and / or LRP protein into a cell, or neuronal cell, but this can be achieved by microinjection or other carrier-mediated methods, or protein delivery or transduction systems (cells (Including Lindsay 2002) with penetrating peptides, ie TAT, transportans, including antennapedia penetrating peptides.

  The present invention allows the generation of a large number of dopaminergic neurons. These dopaminergic neurons can be used as a source material to replace cells that are degenerated or damaged or lost in Parkinson's disease.

  Preferably, the cells are those that undergo mitosis when contacted with a Dkk ligand or Dkk receptor.

  In the methods of the present invention, the cells can be further contacted with one or more substances selected from the following agents: basic fibroblast growth factor (bFGF); epidermal growth factor (EGF); and retinoid Activators of the X receptor (RXR), such as the synthetic retinoid analog SR11237 (Gendimenico, GJ 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); members of the Wnt family of ligands such as Wnt-1, -3a and -5a (Catello-Branco et al., 2003); upstream regulators of Wnt such as Msx-1 (Iler 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 β-catenin (Catello-Branco et al. , 2003); or Nurr1 (Wagner et al., 1999). Treating cells with one or more of the above substances according to the present invention increases the proportion of stem, progenitor, or progenitor cells that assume dopaminergic fate, or increases dopaminergic induction or differentiation in neuronal cells. . A method for eliciting dopaminergic fate in accordance with the present invention, or a method for enhancing dopaminergic induction or differentiation in neuronal cells, comprises treating cells with a member of the FGF family of growth factors, such as FGF4, FGF8 or FGF20, for example in a pre-treatment step. Including contact.

  Advantageously, the cell is contacted with two or more of the above agents.

  Instead of or in addition to pretreatment, the cells may be treated with additional factors simultaneously with Dkk and / or LRP treatment.

  The method of the invention (where neuronal fate is induced in stem cells, neural stem cells, progenitor or progenitor cells, or dopaminergic induction or differentiation is enhanced in neuronal cells) comprises detecting a marker of neuronal fate, β-tubulin III (TuJ1) is one marker of neuronal fate (Menezes, JR et al. (1994) J Neurosci 14 (9), 5399-5416). Other neuronal markers include neurofilament and MAP2. If a specific neuronal phenotype is induced, the marker must be specific for that phenotype. In the case of dopaminergic fate, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine transporter (DAT), and dopamine receptors are e.g. by immunoreactivity or in situ hybridization. Can be detected. Tyrosine hydroxylase is a major marker of DA cells. Dopamine and metabolite content and / or release may be detected, for example, by high pressure liquid chromatography (HPLC) (Cooper, JR 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 an indicator of dopaminergic fate. Additional markers include Nurr1, retinal dehydrogenase 1 (Raldh1) or aldehyde dehydrogenase type 2 (AHD-2), GIRK2, Lmx1a / b and Pitx3.

  The marker can be detected by methods known to those skilled in the art. In the detection method, a specific binding member capable of binding to the nucleic acid sequence encoding the marker is used, and the specific binding member is a nucleic acid probe capable of hybridizing to the sequence, or the nucleic acid sequence or a polypeptide encoded by the nucleic acid sequence. An immunoglobulin / antibody domain with specificity for. A specific binding member is labeled such that its binding to the sequence or polypeptide is detectable. A “specific binding member” has particular specificity for the marker and binds to the marker in preference to other chemical species under normal conditions. Alternatively, if the marker is a specific mRNA, it can be detected by binding to a specific oligonucleotide primer and amplifying (eg, by polymerase chain reaction).

Nucleic acid probes and primers can hybridize to the marker under stringent conditions. Appropriate conditions are, for example, when detecting about 80-90% identical marker sequences, overnight hybridization at 42 ° C. in 0.25 M Na ₂ HPO _4, pH 7.2 _, 6.5% SDS, 10% dextran sulfate, and Includes a final wash at 55 ° C in 0.1X SSC, 0.1% SDS. When detecting about 90% or more of the same marker sequence, suitable conditions are: overnight hybridization at 65 ° C. in 0.25M Na ₂ HPO _4, pH 7.2 _, 6.5% SDS, 10% dextran sulfate, and 0.1 × Includes a final wash in SSC, 0.1% SDS at 60 ° C.

  In a further aspect, the present invention provides a neuron (neural cell) made according to any one of the methods disclosed herein. The neuron may have a primitive neuronal phenotype. Neurons are capable of generating multiple distinct neuronal phenotypes. The neuron may have a specific neuronal phenotype. In a preferred embodiment, the neuron has a dopaminergic phenotype.

  A neuron can include a nucleic acid that encodes a molecule having neuroprotective or neuroregenerative effects, the nucleic acid being operably linked to a promoter capable of driving expression of the molecule in the neuron. is there. The promoter can be an inducible promoter, such as a TetON chimeric promoter, which can prevent damaging overexpression. The promoter may be associated with a specific neuronal phenotype, such as the TH promoter, Nurr1 promoter, dopamine transporter promoter, or Pitx3 promoter.

  The encoded molecule is such that its expression makes the neuron independent of its environment, ie, its survival is one or more factors or conditions (eg, factors or conditions in the neural environment in which it is to be transplanted) It can be something independent of existence. As an example, a neuron can include a nucleic acid that encodes one or more molecules having neuroprotective or neuroregenerative effects described below, which nucleic acid can drive expression of the molecule in the neuron. And functionally linked.

  In addition or alternatively, expression of the encoded molecule may function in neuroprotection or nerve regeneration of the cellular environment surrounding the neuron. In this case, the neurons are used in cell and gene combination therapy to deliver molecules with neuroprotective and neuroregenerative effects.

Examples of molecules that have neuroprotective and neuroregenerative effects include:
(i) Neurotrophic factor. Such factors can compensate and prevent neurodegeneration. One example is glial-derived neurotrophic factor (GDNF), which is a powerful neuronal survival factor that promotes budding from dopaminergic neurons and increases the expression of tyrosine hydroxylase (Tomac et al., (1995). ) Nature, 373, 335-339; Arenas et al. (1995) Neuron, 15, 1465-1473). By increasing axon elongation, GDNF can increase the ability of neurons to distribute nerves in the local environment. Other neurotrophic molecules of the GDNF family include Neurturin, Persephin, and Artemin. The neurotrophin family of neurotrophic molecules includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3, -4/5, -6. Other factors with neurotrophic activity include FGF family members, eg FGF2, 4, 8, 20; Wnt family members, eg Wnt-1, -2, -5a, -3a, 7a; BMP family members For example, BMP2, 4, 5, 7, nodal, activin, GDF; and members of the TGFα / β family.

(ii) anti-apoptotic molecules. Bc12 plays a central role in cell death. Overexpression of Bc12 protects neurons from naturally occurring cell death and ischemia (Martinou et al. (1994) Neuron, 1017-1030). Another anti-apoptotic molecule specific for neurons is BclX-L.

(iii) Axonal regeneration and / or elongation and / or guidance molecules. Such molecules assist neurons in the distribution of nerves and the formation of connections to their environment, such as ephrins. Ephrins define a class of membrane-bound ligands that can activate tyrosine kinase receptors. Ephrins are involved in the development and development of nerves (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 Yue et al. (1999) J Neurosci 19 (6), 2090-2101).

(iv) Transcription factors such as homeobox domain protein Ptx3 (Smidt, MP et al. (1997) Proc Natl Acad Sci USA, 94 (24), 13305-13310), Lmx1a / b, Pax2, Pax5, Pax8, or engrailed 1 Or 2 (Wurst and Bally-Cuif, 2001; Rhinn and Brand, 2001); upstream regulators of Wnt, such as Msx-1 (Iler and Abate-Shen, 1996; Shang et al., 1994); β-catenin (Catello-Branco et al. , 2003); Nurr1 (Wagner et al., 1999); A neurogenic gene of the basic helix loop helix family.

  Neurons according to the invention or neurons used in the invention are substantially free of one or more other cell types, such as stem cells, neural stem cells, progenitor cells or progenitor cells. Neurons can be isolated from neural stem cells or progenitor cells using methods known to those skilled in the art, including methods based on the recognition of extracellular epitopes by magnetic beads and antibodies or fluorescence activated cell sorting (FACS). included. As an example, antibodies against extracellular regions of molecules that are present on stem cells, neural stem cells, progenitor cells or progenitor cells but not on neurons can be utilized. Such antibodies include Notch 1, CD133, SSEA1, prominin 1/2, RPTPβ / phosphocan, TIS21, and glial cell line-derived neurotrophic factor receptor GFRα or NCAM. Stem cells associated with the antibody can be lysed by exposure to complement and can be isolated, for example, by magnetic sorting (Johansson et al., (1999) Cell, 96, 25-34). When using an antibody that is heterologous to the intended neuronal recipient, stem cells, neural stem cells, progenitor cells or progenitor cells that escape such cell sorting methods are labeled with the xenograft antibody, The primary target of the transplanter's immune system. Alternatively, cells that acquire the desired phenotype could be separated by antibodies against extracellular epitopes or by expression of a transgene (such as a fluorescent protein) under the control of a cell type specific promoter. As an example, dopaminergic neurons will be isolated by fluorescent proteins expressed under the control of TH, DAT, Ptx3, or other promoters specifically used by dopaminergic neurons.

  The methods of the invention can include additional negative or positive selections to enrich for neural stem cells, progenitor or progenitor cells, or other stem cells, or neurons with the desired phenotype.

  It is possible to enrich DA neurons using negative selection. Selective neurotoxins for non-DA neurons are used, for example, after addition of 5,7-dihydroxytryptamine (which eliminates serotonergic neurons), or antibodies saponin or toxin coupled, or complement, eg GABA Use antibodies against the transporter, which eliminates GABAergic neurons. The method of the invention comprises further treating or contacting neural stem cells, primordial or progenitor cells, or other stem or neural cells with a negative selection agent, preferably in vitro, for example, This is done by adding a negative selection agent to the in vitro culture containing the cells or by culturing the cells in the presence of the negative selection agent. The negative selection agent selects a cell type other than the desired cell type. For example, when the present invention relates to the promotion, enhancement, or induction of a dopaminergic neuron phenotype, the negative selection agent is DA neurons and cells that develop into DA neurons (eg, stem cells, neural stem cells, progenitor or progenitor cells etc. ) Cells can be selected. Thus, the negative selection agent selects for differentiated cells with a non-DA phenotype, such as non-DA neurons. The negative selection agent suppresses or prevents the growth of cells other than the desired cell type and / or kills cells other than the desired cell type. 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 can be 5,7-dihydroxytryptamine (decreases serotonergic neurons). The negative selection agent may be an antibody or antibody fragment specific for non-DA neurons, in which case the antibody or antibody fragment (eg, scFv or Fab) is a blocking antibody or coupled to a saponin or toxin Is done. For example, such antibodies are blocking antibodies to FGF-4 (decrease serotonin neurons) or antibodies specific for GABA transporters (decrease GABAergic neurons) coupled to toxins.

  In the methods of the present invention, neural stem cells, progenitor or progenitor cells, or other stem or neuronal cells are treated with antioxidants (eg ascorbic acid), hypoxic partial pressure and / or hypoxia inducing factors (eg HIF or erythropoietin). ) In the presence of

  The present invention further includes, in various aspects and embodiments, Dkk ligands and / or Dkk receptors (eg, Dkk polypeptides and / or LRP polypeptides), or nucleic acids encoding Dkk ligands or Dkk receptors, or synthetic From Dkk ligands and / or Dkk receptor analogues, or proteins, nucleic acids or synthetic drugs that act to enhance, switch or modulate one or more signaling components downstream of Dkk and / or LRP Use of the selected agent in the following methods of treatment is provided. The method of treatment induces, promotes or enhances the development of dopaminergic neurons in the brain by acting on endogenous stem cells, progenitor or progenitor cells, or neuronal cells, or exogenously supplied cells And / or in the treatment of individuals suffering from, for example, Parkinson's syndrome or Parkinson's disease, suppressing or preventing the loss of dopaminergic neurons, or survival, phenotypic differentiation, maturation, nerves of dopaminergic neurons Dkk ligands and / or Dkk receptors (eg, Dkk and / or LRP polypeptides), or encoding nucleic acids, or other actions as described above to promote process formation, synaptogenesis, neurotransmission, or functional output Administering the substance to the individual. A Dkk ligand and / or Dkk receptor (eg, Dkk and / or LRP polypeptide), or encoding nucleic acid, or other agent as described above, can be prepared in a suitable composition (eg, a pharmaceutically acceptable excipient or Containing the carrier) and used in the manufacture of a medicament for the treatment of neurodegenerative diseases, Parkinsonism, or Parkinson's disease. Dkk ligands and / or Dkk receptors (eg, Dkk and / or LRP polypeptides), or encoding nucleic acids, can be administered to or targeted to the central nervous system and / or brain.

  The present invention, in various embodiments, encompasses not only neurons generated by any of the methods disclosed herein, but also the following: Pharmaceutical compositions comprising such neurons, stem, progenitor or progenitor cells Product, medicament, drug or other composition; use of such a neuron, stem, progenitor or progenitor cell, or composition in a medical treatment method; such neuron, stem, progenitor or progenitor cell, neuronal cell, or A method comprising administering the composition to a patient, eg for the treatment of Parkinson's disease or other (eg neurodegenerative) diseases (including prophylactic treatment); for such neurons or cells, eg Parkinson's disease or Use in the manufacture of a composition to be administered for the treatment of other (eg neurodegenerative) diseases; May comprise cells together with pharmaceutically acceptable excipients, vehicles or carriers, optionally with one or more other components (eg, neuroprotective molecules, nerve regeneration molecules, retinoids, growth factors, astrocytes / glia cells, anti-cells, A method of producing a pharmaceutical composition comprising mixing with an apoptotic factor, or a factor that modulates gene expression in stem, progenitor or progenitor cells, neuronal cells, or in the host brain. These optional components can make a neuron independent of its environment, ie, its survival is independent of the presence of one or more factors or conditions in the environment. By way of example, a method for producing a pharmaceutical composition includes mixing neurons with one or more factors found in the developing ventral midbrain. Neurons may be mixed with GDNF and / or neurturin (NTN).

  The present invention provides a composition containing neurons, stems, progenitor or progenitor cells, or neuronal cells made according to the present invention, and one or more additional components. A pharmaceutical composition according to the present invention for use according to the present invention comprises, in addition to the aforementioned neurons or cells, a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabilizer, antioxidant, or Other materials well known to those skilled in the art can be included. Such substances must be non-toxic and should not interfere with neuronal activity. The specific nature of the carrier and other materials will depend on the route of administration. The composition comprises one or more of a neuroprotective molecule, a nerve regeneration molecule, a retinoid, a growth factor, an astrocyte / glia cell, or a factor that regulates gene expression in a stem, neural stem, progenitor or precursor cell, or neuronal cell. Can be included. Such substances make the neuron independent of its environment as described above.

  Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Saline solutions, tissue or cell culture media, dextrose or other sugar solutions, or glycols (ethylene glycol, propylene glycol, polyethylene glycol, etc.) may be included.

  The composition may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has an appropriate pH, isotonicity and stability. A person skilled in the art will be able to prepare a suitable solution using, for example, sodium chloride, Ringer's solution, or lactated Ringer's solution. Artificial cerebrospinal fluid may be used to prepare the composition.

The present invention encompasses the use of neurons made according to the present invention in a method of medical treatment, particularly the treatment of medical conditions associated with neuronal cell degeneration, damage, reduction, or disorder. Furthermore, the present invention provides the use of neurons with a particular phenotype in the treatment of symptoms, diseases or disorders associated with degeneration, damage or reduction of neurons of that phenotype. More particularly, the present invention provides the use of dopaminergic neurons in the treatment of human Parkinson's disease. The present invention particularly relates to, but is not limited to, materials and methods for treating neurodegenerative diseases (eg, α-synuclein disorders such as Parkinson's disease). As an example, the present invention treats pathologies or diseases affecting dopaminergic neurons, and degeneration or damage of other areas of the nervous system, particularly areas containing Nurr1 ⁺ cells such as the spinal cord and / or cerebral cortex. It extends to.

  In a method of treatment where the cells being administered are stem cells, progenitor or progenitor cells capable of producing two or more different neuronal phenotypes, the neuron, cell or composition is treated with a region comprising astrocytes / glia cells ( The cell differentiation can be introduced into the desired specific neuronal fate). The cell or composition is, for example, injected into the ventral mesencephalon where it is induced to interact with type 1 astrocytes / glia cells and adopt a dopaminergic phenotype. Alternatively or in addition, the composition to be transplanted may contain one or more factors (eg, type 1 astrocytes / cells) that direct the development of neurons or cells towards a specific neuronal fate as described above. In combination with glial cells).

  Cells can be transplanted into a patient by techniques known in the art (eg, Lindvall, O., (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, CR, (1992) New England Journal of Medicine, 327, 1549-1555).

  Administration of the composition according to the invention is preferably carried out in a “prophylactically effective amount” or “therapeutically effective amount” (although in some cases prophylaxis is considered a treatment), this may benefit the individual. Enough to bring. The actual dosage, and rate and time course of administration, will depend on the nature and severity of the person being treated. The prescription for treatment (eg, determination of dosage) is within the discretion of the practitioner or other physician.

  The composition is administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition to be treated.

  The methods provided herein can be practiced using primary cells or cell lines in vivo or in vitro as raw materials. The advantage of growing cells in vitro is that there is virtually no limit on the number of neurons produced.

  In order to reduce possible disadvantages associated with immune rejection of transplanted cells, stem, progenitor or progenitor cells are isolated from the patient and induced to the desired phenotype. The cells are then transplanted into the patient. Advantageously, cell lines are established using isolated stem cells, progenitor or progenitor cells to obtain a large number of immunocompatible neuronal cells. A further option is to establish a cell bank that covers a range of immunocompatibility from which cells suitable for an individual patient can be selected. Stem cells, neural stem cells, progenitor or precursor cells, or neuronal cells from one individual can be modified to reduce or prevent rejection when the cell or its progeny are introduced into another individual. As an example, one or more MHC alleles of a donor cell are replaced with that of the recipient, for example by homologous recombination.

  When cells derived from a cell line carrying an immortal oncogene or reporter gene are used for transplantation into a patient, the oncogene may be removed by the CRE-loxP system before transplanting the cell into the patient. (Westerman, KA et al. Proc. Natl. Acad Sci. USA 93, 8971 (1996)). Immortal oncogenes that are not active at the patient's body temperature may be used.

  In a further aspect, the present invention includes the use of cells or neurons produced according to the present invention in a method for screening a drug for the treatment of neurodegenerative diseases. The neuron can be a dopaminergic neuron. The neurodegenerative disease can be α-synucleinopathy such as Parkinson's syndrome or Parkinson's disease. The agent can be a neuroprotective and / or neuroregenerative molecule and / or a developmental soluble signal and / or one or more factors derived from ventral mesencephalic type 1 astrocytes or glial cells. This method can be performed in vitro or in vivo.

The method can include the following steps:
(i) treating the neuron of the present invention with a toxin of the neuron;
(ii) separating the neurons from the toxin;
(iii) contacting the treated neurons with one or more test agents;
(iv) measuring the ability of the neuron to recover from the toxin;
(v) Compare the ability of the neurons to recover from the toxin with the ability of the same neurons not contacted with the test agent to recover from the toxin.

The method can include the following steps:
(i) treating a neuron of the invention with a toxin of the neuron in the presence of one or more test agents;
(ii) measuring the resistance of the neuron to the toxin;
(iii) Compare the resistance of the neuron to the toxin with that of the same neuron not contacted with the test agent.

  Toxins are 6-hydroxydopamine, 5,7-dihydroxytryptamine, or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), proteasome inhibitors (including lactacystin), Or it may be an insecticide (including rotenone), all of which lead to the death of catecholaminergic neurons and experimentally reproduce the characteristics of Parkinson's disease. The ability of neurons to recover from toxins or resistance to toxins can be measured by methods known to those skilled in the art, such as cell viability or cell loss (eg by cell count, eg by TUNEL method) or active caspase -3 Measured by monitoring immunostaining, monitoring morphology (eg germination, axon elongation and / or branching), and / or monitoring biochemistry (eg TH activity, eg neurotransmitter uptake / release / content) To do.

A method of screening for a drug for treating a neurodegenerative disease can include the following steps:
(i) contacting dopaminergic neurons produced by the above method with one or more test agents;
(ii) confirming the growth, function, maintenance, viability or neurotransmitter release of the dopaminergic neurons in the presence of the drug.

In another embodiment, a method for screening a drug for treating a neurodegenerative disease utilizes neurons generated from stem, progenitor or progenitor cells under oxidative stress and comprises the following steps:
(i) Prepare neurons derived from stem cells, progenitor or progenitor cells under oxidative stress,
(ii) contacting the neuron with one or more test agents;
(iii) confirming the growth, function, maintenance, viability or neurotransmitter release of the neuron in the presence of the agent.

  Neuronal growth, function, maintenance, viability or neurotransmitter release in the presence of the drug is defined as neuronal growth, function, maintenance, viability or neurotransmitter release in the absence of the drug. Can be compared. An increase in growth, neurotransmission, maintenance, or viability in the presence of a drug compared to the absence of the drug will be useful in the treatment of a neurodegenerative disease.

Neuronal growth, neurotransmission, maintenance, or viability can be measured by methods known to those skilled in the art, for example, BrdU or ³ by cell count (eg, by TUNEL method) or active caspase-3 immunostaining. By H-thymidine uptake, by monitoring morphology (eg germination, axon elongation and / or branching), by biochemistry (eg TH activity, eg neurotransmitter uptake / release / content) and / or It can be measured by gene or protein expression (eg, immunohistochemistry, PCR, gene chip, proteome).

  Examples of stem cells, neural stem cells, embryonic stem cells, progenitor cells, progenitor cells, or neurons that can be used in the present invention include C17.2 (Snyder, EY et al. Cell 68, 33-51 (1992)) and H6 human cells. Strains (Flax et al. Nature Biotech 16 (1998)). Additional examples of human neural and embryonic stem cells are Gage, FH et al. Ann. Rev. Neurosci. 18 159-192 (1995); Gotlieb (2002) Annu. Rev. Neurosci 25 381-407); Carpenter et al. Stem Cells. 5 (1): 79-88 (2003); and http://stemcells.nih.gov/research/registry/.

  Although discussed herein with respect to neural stem cells or neural progenitor or progenitor cells, the methods provided herein can also be applied to induce neuronal fate in other stem cells, progenitor or progenitor cells. Examples of such cells include human and non-human embryonic stem cells, and stem cells associated with the non-neural system. The method can be applied to stromal cells or hematopoietic stem cells and / or epithelial proliferative cells. Hematopoietic cells can be collected from blood or bone marrow biopsy. Stromal cells can be collected from a bone marrow biopsy. Epithelial cells can be collected by skin biopsy or by rubbing the oral mucosa, for example. Because the neuronal phenotype is not the physiological fate of these stem cells, progenitor or progenitor cells in vivo, this induction method is called trans-differentiation, or de-differentiation and neural redifferentiation It is. Methods for inducing such cells to neuronal fate use microRNAs or anti-sense regulators for genes associated with non-neuronal phenotypes to suppress and / or differentiate these cells into non-neuronal fate. Or including flipping.

  The method of the present invention can be applied to stem cells that are not tied to neural fate. They can be applied to stem cells that are capable of generating two or more daughter stem cells associated with different developmental lines. Examples of such stem cells are embryonic stem cells, hematopoietic stem cells, epithelial proliferative cells, and neural stem cells.

  In various further embodiments, the present invention enhances induction of dopaminergic fate in neural stem cells or progenitor or progenitor cells, or with Dkk ligands (eg, Dkk polypeptides) and / or Dkk receptors (eg, LRP polypeptides). It relates to providing assays and screening methods for one or more factors that enhance dopaminergic induction or differentiation in treated neuronal cells.

  The present invention can enhance, increase or enhance induction of dopaminergic fate in stem cells, neural stem cells, progenitor or precursor cells, or neuronal cells, alone or in combination, in the presence of Dkk and / or LRP Methods of screening for one or more factors are provided. A further aspect of the invention is a screening for obtaining and / or identifying one or more factors that enhance the induction of dopaminergic fate in stem cells, embryonic stem cells, neural stem cells, progenitor or precursor cells, or neuronal cells or Provided is the use of stem cells, embryonic stem cells, neural stem cells, progenitor or precursor cells, or neuronal cells in the presence of Dkk and / or LRP in searching.

The screening method can include the following steps:
(a) contacting the test substance with stem cells, neural stem cells, progenitor or progenitor cells or neuronal cells in the presence of a Dkk ligand (eg Dkk polypeptide) and / or a Dkk receptor (eg LRP polypeptide) The contact can result in an interaction between the test substance and the cell;
(b) Confirm the interaction between the test substance and the cells.

  The interaction is a signaling pathway or cellular process that results in or accompanies Dkk ligand / receptor polypeptide binding and uptake into the cell, and / or increased dopaminergic cell number and / or differentiation. This can be confirmed by measuring the ability of the cells to activate. These include signaling processes regulated by kremen or LRP 5/6 receptors, i.e. levels and / or degradation and / or translocation and / or phosphorylation of β-catenin or axin as described herein Adjustments are included.

  The screening method is a membrane fraction derived from a test substance from stem cells, neural stem cells, progenitor or precursor cells, or neuronal cells in the presence of a Dkk ligand (eg Dkk polypeptide) and / or a Dkk receptor (eg LRP polypeptide). Contacting the fraction, soluble fraction or nuclear fraction and confirming the interaction between the test substance and the fraction. The preparation of such fractions is within the ability of one skilled in the art.

  Binding or interaction can be confirmed by several techniques (qualitative or quantitative) known in the art. The interaction between the test substance and the stem cells or progenitor cells or neuronal cells can be investigated by attaching a detectable label to one of them and bringing it into contact with the other, for example the solid phase support It may be immobilized on a solid support using antibodies bound to the body or by other techniques known per se, including the Biacore system.

  Screening methods involve culturing stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells in the presence of one or more test substances, and for the differentiation of the cells into a dopaminergic phenotype (eg, as described herein). Analyzing) by detecting markers of the described dopaminergic phenotype. One of the markers of dopaminergic phenotype is tyrosine hydroxylase (TH). Additional markers include Nurr1, retinal dehydrogenase 1 (Raldh1) or aldehyde dehydrogenase type 2 (AHD-2), GIRK2, Lmx1a / b and Pitx3. The absence of dopamine β-hydroxylase and GABA or GAD (in the presence of TH / dopamine / DAT) is also an indicator of dopaminergic fate.

  Substances screened by the present invention are natural or synthetic compounds, including, for example, polypeptides, nucleic acids, small organic molecules.

  Screening methods can induce dopaminergic fate in ventral mesencephalic type 1 astrocytes or early glial cells in stem cells, neural stem cells, or progenitor or progenitor cells in the presence of Dkk and / or LRP polypeptides Comparing with incapable nerve cells (eg astrocytes). Such a comparison can be made, for example, between developing type 1 astrocytes or early glial cells in the ventral mesencephalon and type 1 astrocytes or early glia from other neural sites.

  In screening methods that require astrocytes or early glial cells, immortalized astrocytes or glial cells can be used. It can be an astrocyte cell line or a glial cell line (eg, an astrocyte or glial midbrain cell line). Such cell lines provide a uniform cell population.

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

  In the screening method, a nucleic acid expression array such as a mouse cDNA expression array can be used. In this approach, an array of different nucleic acid molecules is placed on a filter, quartz, or another surface, for example by cross-linking nucleic acids to the filter. A test solution or extract is obtained and the nucleic acid contained therein is labeled, for example, with fluorescence. The solution or extract is then applied to a filter or gene chip. The hybridization between the test nucleic acid and the nucleic acid on the filter or gene chip is confirmed and compared with the hybridization obtained using the control solution. The hybridization differences obtained for the test and control samples indicate different nucleic acid contents. For more information on nucleic acid arrays, see the Clontech website (eg www.clontech.com) or the Affymetrix website (eg www.affymetrix.com), which can be found using available web browsers.

  The screening method comprises stem cells, progenitor or progenitor cells, or neurons, stem cells, progenitor or progenitor cells in the presence of Dkk ligand (eg, Dkk polypeptide) and / or Dkk receptor (eg, LRP polypeptide), Or, for example, identifying one or more factors compared to a neuronal cell. Such factors enhance cell proliferation and / or self-renewal and / or differentiation and / or survival in the presence of Dkk ligand or Dkk receptor and / or promote acquisition or induction of dopaminergic fate and / or Alternatively, it induces neuronal development and / or neurotransmission in stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells and / or enhances dopaminergic induction or differentiation in neuronal cells. Once the target genes and / or factors have been identified, they can be isolated and / or purified and / or cloned and used in further methods.

  The screening method involves culturing stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells in the presence of one or more test substances, and culturing the cells for β-catenin activation, such as dephosphorylation as described herein. It may comprise analyzing by an oxidation or reporter assay.

  The screening method comprises stem cells, progenitor or progenitor cells, or neurons, stem cells, progenitor or progenitor cells in the presence of Dkk ligand (eg, Dkk polypeptide) and / or Dkk receptor (eg, LRP polypeptide), Or one or more that promotes, increases or enhances β-catenin activation in stem cells, neural stem cells, progenitor or precursor cells, or neurons, for example, in the presence of Dkk ligand or Dkk receptor compared to neurons Identifying the factors. Once the target genes and / or factors have been identified, they can be isolated and / or purified and / or cloned and used in further methods.

  In a related aspect, the invention provides a Dkk ligand or Dkk receptor (eg, Dkk peptide or LRP polypeptide) that induces dopaminergic fate or activates β-catenin in stem cells, neural stem cells, progenitor or progenitor cells. Methods of screening for substances that modulate the ability of are provided.

  Thus, the method comprises a Dkk ligand or stem cell, neural stem cell, progenitor or progenitor cell, or Dkk ligand that induces proliferation, self-replication, dopaminergic development, differentiation, maturation, and / or acquisition of dopaminergic fate, or Agents that modulate the ability of the Dkk receptor (eg, Dkk peptide or LRP polypeptide) can be screened.

Such a method comprises the following steps:
(i) stem cells, embryonic stem cells, neural stem cells, progenitor or progenitor cells in the presence of Dkk ligands and / or Dkk receptors (eg, Dkk peptides and / or LRP polypeptides) and one or more test substances, or Prepare nerve cells,
(ii) analyzing the proportion of cells that take dopaminergic fate or phenotype and / or respond to Dkk ligand and / or Dkk receptor;
(iii) the proportion of cells that take such a dopaminergic fate is determined by taking a dopaminergic fate or phenotype and / or Dkk ligand and / or in the absence of the test substance under comparable reaction medium and conditions Compare with the number of cells that respond to the Dkk receptor. The difference in the proportion of dopaminergic neurons between treated and untreated cells indicates the modulating effect of the relevant test substance.

Such screening methods can include the following steps:
(i) in the presence of one or more test substances, stem cells, embryonic stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells are treated with Dkk ligands and / or Dkk receptors (eg Dkk peptides and / or LRP poly Peptide),
(ii) analyzing the proportion of stem cells, embryonic stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells that adopt dopaminergic fate or phenotype and / or respond to Dkk ligand and / or Dkk receptor;
(iii) a response comparable to the proportion of stem cells, embryonic stem cells, neural stem cells, progenitor or progenitor cells, or neurons that adopt dopaminergic fate or phenotype and / or respond to Dkk ligand and / or Dkk receptor Compare to the number of stem cells, progenitor or progenitor cells, or neurons that take dopaminergic fate or phenotype and / or respond to Dkk ligand and / or Dkk receptor under conditions but in the absence of the test substance To do.

In other embodiments, the screening method can include the following steps:
(i) in the presence of one or more test substances, stem cells, embryonic stem cells, neural stem cells, progenitor or progenitor cells, or neuronal cells are treated with Dkk ligands and / or Dkk receptors (eg Dkk peptides and / or LRP poly Peptide),
(ii) analyzing the proportion of stem cells, embryonic stem cells, neural stem cells, progenitor or precursor cells, or neuronal cells that undergo β-catenin activation in response to Dkk ligands and / or Dkk receptors;
(iii) The proportion of stem cells, embryonic stem cells, neural stem cells, progenitor or precursor cells, or neuronal cells that undergo β-catenin activation in response to Dkk ligands and / or Dkk receptors, under comparable reaction conditions Is compared to the number of stem cells, progenitor or progenitor cells, or neurons that undergo β-catenin activation in the absence of the test substance.

  Such screening methods can be performed on cells in vivo in similar or identical non-human animals or in vitro, ie in culture.

  As mentioned above, substances to be screened according to the present invention may be natural or synthetic compounds such as polypeptides, nucleic acids, small organic molecules.

In other embodiments, dopaminergic by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and / or maturation in stem cells, embryonic stem cells, neural stem cells, progenitor or progenitor cells Methods for screening for compounds useful for inducing or promoting neuron development include:
Measuring the ability of the precursor protein convertase to proteolytically cleave the Dkk polypeptide in the presence of the test compound.

  An increase or decrease in the ability in the presence relative to the absence of the test compound indicates that the test compound is useful for inducing or promoting the development of dopaminergic neurons.

  The ability of a precursor protein convertase to proteolytically cleave a Dkk polypeptide is measured by measuring Dkk activity (eg, by measuring or detecting β-catenin activation or dopaminergic development), or by Dkk polypeptide It can be measured by confirming the presence of the hydrolyzate.

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

  The precursor protein convertase may be a protease specific for the sequence Arg-XX-Arg, Arg / Lys-XXX-Arg / Lys-Arg, or Arg / Lys-Arg, for example furin or protease convertase 2 (PC2 ).

  In other embodiments, a method for screening a Dkk ligand that is not prone to proteolysis comprises confirming the Dkk activity of a sample that is expected to contain a Dkk ligand in the presence of a proprotein convertase, wherein the activity is present. In this case, the sample contains the Dkk ligand.

  The Dkk activity of the sample is increased in dopaminergic neurons by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and / or maturation in embryonic stem cells, neural stem cells, progenitor or precursor cells By measuring the effect of the sample on promoting development, measuring the effect of the sample on β-catenin activation in embryonic stem cells, neural stem cells, progenitor or progenitor cells, or Dkk ligand protein This can be confirmed by confirming the presence or absence of a product of degradation cleavage.

  One method can include identifying a sample containing a Dkk ligand that is not prone to proteolysis. The Dkk ligand may be isolated and / or purified from the sample.

  Screening methods can include isolating and / or purifying one or more substances from a mixture or sample. This method involves the use of stem cells, neural stem cells, neural progenitor or progenitor cells, or mixtures that interact with neural cells in the presence of Dkk ligands (eg, Dkk polypeptides) and / or Dkk receptors (eg, LRP polypeptides). The ability of one or more fractions (eg, the ability to bind to the cell and / or promote the proliferation and / or self-renewal of the cell, and / or the dopaminergic phenotype and / or nerve in the cell) Measuring the ability to increase the induction, acquisition, differentiation or development of transmission or fate). Any method known to those skilled in the art may be used for purification and / or isolation.

  In the screening method, an inducible promoter operably linked to a nucleic acid encoding a test substance can be used. Such a construct is incorporated into a host cell and one or more properties of the cell under permissive and non-permissive conditions of the promoter are examined and compared. Properties considered are dopaminergic in stem cells, neural stem cells, neural progenitor or progenitor cells, or neurons in the presence of Dkk ligands (eg, Dkk polypeptides) and / or Dkk receptors (eg, LRP polypeptides) Or the ability of the host cell to induce β-catenin activation. If there is a difference in the ability of the host cell under permissive and non-permissive conditions, the test substance, alone or in combination, in the presence of Dkk ligand and / or Dkk receptor, stem cells, neural stem cells, progenitors Or can increase proliferation and / or self-renewal and / or dopaminergic fate and / or induction of dopaminergic differentiation or neurotransmission, survival or development in progenitor cells, or dopaminergic in neuronal cells Indicates that induction or neurotransmission or differentiation can be enhanced.

  Those skilled in the art will be able to change any screening method format of the present invention using ordinary skill and knowledge. Any substance to be tested for modulating activity may be a natural or synthetic chemical.

  A compound, substance, or factor identified by any of the methods provided by the present invention can be isolated and / or purified and / or further studied. It may be manufactured and used to prepare (ie, manufacture or formulate) a composition such as a drug, pharmaceutical composition, drug.

  In various further aspects, the present invention provides an agent identified by any of the methods disclosed herein; a pharmaceutical composition, agent, drug or other composition comprising such an agent (such a composition is a stem cell). , Neural stem cells, progenitor or progenitor cells, or neurons may be included with Dkk ligands (eg, Dkk polypeptides) and / or Dkk receptors (eg, LRP polypeptides); Dkk ligands (eg, Dkk polypeptides) and / or Or induction and / or phenotypic differentiation or maturation and / or survival of dopaminergic neurons derived from stem cells, neural stem cells, embryonic stem cells, progenitor or progenitor cells in the presence of Dkk receptors (eg LRP polypeptides) And / or increase nerve regeneration and / or synaptic regeneration and / or neurotransmission and / or functional output Use of the factor or composition in a medical treatment method; medical condition associated with degeneration, damage, reduction or disorder of dopaminergic neurons, or medical effects on dopaminergic neurons A method comprising administering the factor or composition to a patient for treating a symptom (including prophylactic treatment), eg for treating Parkinson's disease or other neurodegenerative disease; for administration, eg Parkinson Use of the factor in the manufacture of a composition, medicament or drug for the treatment of a disease or other (such as a neurodegenerative disease); and the factor with a pharmaceutically acceptable excipient, vehicle or carrier and optionally other There is further provided a method of preparing a pharmaceutical composition comprising combining with the ingredients of

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

  The present invention encompasses all combinations and subcombinations of features described above.

  Several aspects and embodiments of the invention are illustrated by way of example and are described with reference to the drawings described below.

  Figures 1 to 6 show the continuous expression of Dkk-1, -2, -3 in the developing VM (ventral mesencephalon). Statistical analysis was performed using one-way analysis of variance (One-Way ANOVA) and Fisher's post-hoc test.

  Specifically incorporated herein by reference are the experimental results described in WO 00/667136 and WO 2004/029229. WO 00/667136 describes the proliferation and / or self-renewal of dopaminergic progenitor cells and dopaminergic activity in stem cells, neural stem cells, progenitor cells or progenitor cells that express Nurr1 in the presence of type 1 astrocytes or glial cells It demonstrates the induction of neurons and the additional results obtained when the cells are contacted with additional factors such as FGF (eg FGF8) or retinoids. And WO 2004/029229 describes the proliferation and / or self-renewal of dopaminergic progenitor cells and the expression of NG4A subfamily nuclear receptors such as Nurr1 in cells beyond the basal level, Demonstrates the induction of dopaminergic neurons in stem cells, neural stem cells, progenitor cells or progenitor cells upon treatment.

Partial purification of conditioned medium containing Dkk-AP and Dkk-FLAG, and instructions for using furin inhibitor-treated human embryonic kidney cells 293T (HEK293T) cells or SN4741 cells using Lipofectamine 2000 (Lipofectamine 2000: Invitrogen) And transiently transfected with the Dkk-AP (Mao et al., 2001a) plasmid, or the full-length or truncated Dkk-FLAG (Li et al., 2002) plasmid. After 48 hours, serum-free N2 conditioned medium was collected and concentrated using a Centricon-Plus 20 column (Millipore) according to the instructions for use. The protein content was measured and aliquots (aliquots of samples) of equal concentration were prepared and stored at -80 ° C. For furin inhibition experiments, DMSO (as a control) or decanoyl-RVKR-CMK (Calbiochem) was added to the culture 4 hours after the start of transfection and OPTIMEM medium (Invitrogen) was replaced with N2 medium. cm (conditioned medium) was collected after 24 hours, centrifuged to remove any cellular contaminants and analyzed directly (no pp (partial purification)).

The embryonic (E) 14.5 day ventral mesencephalon (VM) obtained from a Sprague-Dawley rat mated with a progenitor cell culture on a regular basis. Were removed and mechanically dissociated. It was then made of a 1: 1 mixture of F12 and MEM (Invitrogen) containing 15 mM HEPES buffer and 1 mM glutamine (Invitrogen) in a poly D-lysine (10 μg / ml) coated plate (Falcon). Serum N2 medium was seeded at a final density of 1 × 10 ⁵ cells / cm ² . N2 supplement and 5 mg / ml AlbuMAX (provided by Invitrogen) were also added to the medium. Alternatively, add 5 μg / ml insulin, 100 μg / ml apotransferrin, 60 μM putrescine, 20 nM progesterone, 30 nM selenium, 6 mg / ml glucose, and 1 mg / ml BSA (all purchased from Sigma) did. Cultures were cultured for 3 days in vitro under 5% CO ₂ in a 37 ° C. incubator before fixation. Recombinant mouse Dkk-1 (R & D Systems) and recombinant human Dkk-2 (AmProx, USA) were used at final concentrations of 0-500 ng / ml. For Dkk-AP and truncated Dkk-FLAG, 25 μg / μl stock solution was added at 0-10 μg / ml. All these reagents were added to the progenitor cell culture at the start of the culture.

  For Dkk-AP and LRP-6N-Fc, 25 μg / μl stock solution was added at 0-10 μg / ml. All these reagents were added to the progenitor cell culture at the start of the culture.

Glial culture glial cultures (O'Malley et al., 1992) were obtained from postnatal day 1 (P1) VM, cortex (Cx), and striatum (S1) from Sprague-Dawley rats mated regularly. Established from Str). After mechanical dissociation, 10 × 10 ⁶ cells were added to NM15 medium (15% fetal calf serum, 7.2 mg / ml glucose, 2.4 in MEM in a 25 cm ² flask previously coated with 10 μM poly D-lysine. Seeded in mM glutamine, 0.5% Fungizone, and 0.6% gentamycin (all purchased from Sigma). Cells were grown to a monolayer and microglia and cell debris were removed by shaking at 190 rpm for 1 hour at 37 ° C. Then, the adherent cells were washed with sterile PBS, cultured in 5% CO _{2 for} 1-2 hours in a 37 ° C. incubator, and then shaken overnight at 37 ° C. at 210 rpm. Thereafter, the supernatant medium containing oligodendrocyte progenitor cells and other cell types was discarded. The remaining glial monolayer was then dissolved for RNA recovery.

Real-time PCR and gene expression quantification
Primers were designed with Primer Express 2.0 (PE Applied Biosystems, Foster City, California, USA) using the GenBank cDNA sequence. The specificity of the PCR primer was determined by searching the primer sequence with BLAST. The oligonucleotides listed in Table 1 were used. All primers except Quantum RNA classical 18S internal standard (Ambion, Austin, USA) were purchased from DNA Technologies (Denmark).

  RNeasy extraction kit (Qiagen, from the ventral midbrain pool excised from E10.5, E11.5, E13.5, E15.5, and P1 rats and from glial monolayers (n = 4 for each region) Total RNA was isolated using Hilden, Germany). For reverse transcription reaction, 1 μg of total RNA was first treated with 1 unit of RQ1 RNase-Free DNase (Promega, Madison, USA) for 40 minutes. 1 μl of 0.02 M EDTA was added and incubated at 65 ° C. for 10 minutes to inactivate DNase. 0.5 μg of random primer (Invitrogen) was then added and the mixture was incubated at 65 ° C. for 5 minutes. Each sample was then aliquoted into two tubes, a cDNA reaction tube and a negative control tube. Thereafter, a master mixture containing 1 × First-Strand buffer (Invitrogen), 0.01M DTT (Invitrogen) and 0.5 mM dNTP (Promega, Madison, USA) was added to both cDNA and RT-tubes at 25 ° C. Incubated for 10 minutes. Subsequently, it was incubated at 42 ° C. for 2 minutes. Supercript II reverse transcriptase (200 units, Invitrogen) was then added only to the cDNA tube and all samples were incubated at 42 ° C. for 50 minutes. Supercript II was inactivated by incubating at 70 ° C. for 10 minutes. Subsequently, both cDNA and RT- were diluted 10-fold for further analysis.

Real-time RT-PCR reactions were performed in 3 master mixes containing 1 μl of 10 × diluted cDNA or 10 × diluted RT− per 25 μl final volume. Each PCR reaction solution consists of 1x PCR buffer (Invitrogen), 3 mM MgCl ₂ (Invitrogen), 0.2 mM dNTP (Promega, Madison, USA), 0.3 µM forward (forward) and reverse (reverse) Consists of primers, 0.5 units Platinum Taq DNA polymerase (Invitrogen), and 1 × SYBR Green (Molecular Probes, Leiden, The Netherlands). PCR was performed at 94 ° C for 2 minutes, followed by 35-40 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 15 seconds and 80 ° C for 5 seconds (for SYBR Green detection), ABI PRISM Performed on a 5700 detection system (PE Applied Biosystems, Foster City, California, USA). To confirm that the SYBR Green signal corresponds to a unique and specific amplicon, a melting curve was obtained for each PCR product after each reaction. Use a serial 3-fold dilution of a control sample containing the sequence of interest (reverse transcribed RNA, contained in a plasmid, or genomic DNA) for each probe in every 96-well plate for each real-time PCR A standard curve was created. The resulting standard curve graph was then used to convert Cts (the number of PCR cycles required to amplify a given template to an established fluorescence threshold) into an arbitrary amount of the initial template for a given sample. The expression level is obtained by subtracting the corresponding RT− value from the RT + value of each sample (if appropriate) and then dividing that number by the value of the housekeeping gene 18S obtained for each sample in a parallel assay. Each assay was repeated twice for a particular gene. The 18S assay was performed once or twice for each sample at the beginning and in the middle of the assay to verify sample integrity.

  PCR products were run on a 2% agarose gel to verify amplicon size. Statistical analysis of the results was performed by one-way analysis of variance. This was followed by Fisher's protected least significant difference to identify specific points whose different stages of development differed from the original only when significant interactions occurred. Significance of all tests was estimated at a level of p <0.05 (* p <0.05; ** p <0.001; *** p <0.0001).

in situ hybridization
Wild-type and Lrp6 mutant mouse embryos at E11.5, E12.5, and E15.5 were fixed overnight at 4 ° C. with 4% paraformaldehyde and embedded in paraffin. 8 μm serial sections were processed for radioactive in situ hybridization using [S35] -UTP labeled antisense riboprobe. Hybridization was performed at 56 ° C. in 50% formamide according to the modified protocol of Dagerlind et al. (Dagerlind et al., 1992). Sections were counterstained with Cresyl Violet (Sigma). Probes for in situ hybridization are as follows. That is, Dkk1, Dkk2 (Monaghan et al., 1999), Lrp5, Lrp6 (PCR products of 2884-3444bp region in Lrp5 of NM_008513 and 2941-3446bp region in Lrp6 of NM_008514), TH, Pitx3 (special gift from Mr. Jordi Guimera) Brodski et al., 2003)), En1 (Davis and Joyner, 1988), Fgf8 (Martinez et al., 1999) and Shh.

The BrdU detection kit (Roche, Germany) was used with a slight modification for the BrdU immunostaining proliferation assay. Two hours before sacrifice, 5-bromo-2 deoxyuridine (BrdU, 1000 μg / 100 g) was injected into the peritoneum of pregnant mice. Embryo / brain was fixed in 4% paraformaldehyde overnight at 4 ° C., dehydrated with ethanol and rotihistol, and then paraffin embedded. Paraffin sections (8 μm) were deparaffinized in rotihistol, rehydrated, and treated with sodium citrate (0.01 M) for 5 minutes. Subsequently, the plate was washed with PBS and incubated in a blocking solution (PBS containing 10% fetal bovine serum and 0.05% Triton X100) for 1 hour. The slides were then incubated overnight at 4 ° C. with the primary antibody anti-BrdU antibody (diluted 1:10 in PBS containing 0.05% Triton X100). After washing 3 times with PBS, the sections were incubated for 2 hours with a secondary antibody, biotinylated anti-mouse antibody (1: 500, Jackson ImmunoResearch, USA). After washing three times with PBS, the slides were incubated with ABC solution (ABC-Kit, VECTASTAIN, Vector Laboratories) for 30 minutes, and diaminobenzidine staining (DAB, Sigma) was performed until a signal could be observed. The slide was washed twice with PBS, dehydrated, and then encapsulated using Roti-Histokit (ROTH, Germany).

Immunocytochemical cells were fixed with ice-cold 4% paraformaldehyde for 20 minutes and then washed with PBS. They were then blocked by incubation with PBT (PBS containing 1% BSA, 0.3% Triton X100, and 0.02% sodium azide, all purchased from Sigma) for 15 minutes. After overnight incubation at 4 ° C. with primary antibodies (mouse anti-TH (1: 1000 dilution, Immunostar), rabbit anti-TH (1: 250, Pelfreeze), rabbit anti-Nurr1 (1: 1000, Santa Cruz)) The cells are washed twice with PBS for 10 minutes and then the appropriate secondary antibody (biotinylated 1: 500 anti-mouse or anti-rabbit IgG (Vector), cyanine-2 or rhodamine-conjugated horse anti-mouse IgG 1: 200, Or incubated with goat anti-rabbit IgG 1: 200 (Jackson Laboratories)) for 2 hours. After washing twice with PBS, the cells were counterstained with Hoechst33258 for 10 minutes (in the case of immunofluorescence) or incubated with avidin-biotin complex solution (Vector) for 2 hours. After washing twice with PBS, the stain was added to the chromogen VIP Vector Laboratories ABC immunoperoxidase kit (red staining: TH) or 3,3 ′ diaminobenzidine tetrahydrochloride (DAB 0.5 mg / ml) / nickel chloride (1.6 The color was developed using (mg / ml) (gray / black: Nurr1).

  Nurr1 staining was performed before TH staining. Images of stained cells in PBS were captured at room temperature using a Zeiss Axioplan 100M microscope (LD Achrostigmat 20 ×, 0.3 PH1∞0-2, and LD Achroplan 40 ×, 0.60 Korr, PH2∞0-2) Captured with Hamamatsu camera C4742-95 (using QED image software). Image processing was performed using Adobe Photoshop version 7.0.

  Quantitative immunocytochemistry data is shown as the mean ± standard error of the mean obtained from 10 non-overlapping fields in 2-3 replicas per condition from 2-3 separate experiments. . Statistical analysis was performed with Stat View (Cary, NC, USA) or Prism 4 software (Graph Pad, San Diego, USA) as described in the figure legends. The significance of all tests was estimated at the level of p <0.05 (* p <0.05; ** 0.01 <p <0.001; *** p <0.001).

Immunoblotting
Partially purified conditioned media from HEK293 cells overexpressing Dkk-AP and LRP-6N-Fc was mixed with 1 × Laemili buffer (1:20). The sample was boiled and subjected to 10% SDS PAGE (15 μl medium sample per lane) and electrotransferred to a Hybond-P PVDF membrane.

  Block the membrane with 5% milk, anti-human placental alkaline phosphatase specific primary antibody (PLAP: Abcam), anti-Dkk-3 specific primary antibody (R & D Systems) and anti-human IgG specific primary antibody (Vector) And immunodetection using the appropriate secondary antibody. The signal was visualized with an ECL + Plus detection system (Amersham Bioscience) according to instructions. After immunodetection, total protein was stained with amide black to confirm equal protein loading.

Immunohistochemistry of LRP-6 mutants
The LRP-6 mutant (LRP-6-/-) (Pinson et al., 2000) was bred, bred and processed according to the ethical approval of animal experiments approved by Stockholms Norra Djurfoersoeks Etiska Naemnd. Wild-type and heterozygous mice were identified by a genotyping PCR reaction method using LRP-6-U1 and LRP-6-D1 primers described previously (Kelly et al., 2004). Furthermore, mice with gene trap insertions were confirmed using the following primer sets. That is, CD4mix forward: 5′-GCACGGATGTCTCAGATCAAGAGG-3 ′ and CD4mix reverse: 5′-CGGGATCATCGCTCCCATATATG-3 ′ having an annealing temperature of 63 ° C. and producing a 108 bp amplicon. Ear or embryonic tissue was boiled in 100-200 μl of 25 mM NaOH / 0.2 mM EDTA for 40 minutes at 95 ° C. and then an equal volume of 40 mM Tris HCl (pH 5) was added. 3 μl of this solution was used for PCR. This PCR was performed as described for real-time PCR, but 30 cycles were performed without SYBR Green and without the 80 ° C. step.

  E11.5, E13.5, and E17.5 mice were fixed overnight with 4% paraformaldehyde and then immersed in a 20% sucrose gradient. Thereafter, the samples were snap frozen at −80 ° C. in O.C.T. Serial sagittal and coronal sections (14 μm thick) were collected on microscope slides (StarFrost) and stored at −80 ° C. Slides were thawed for immunohistochemistry and incubated with 4% paraformaldehyde for 10 minutes. After washing with PBS three times for 15 minutes, the slides were blocked with PBTG (PBT containing 5% goat serum) for 30 minutes. Primary antibodies contained in PBTG (rabbit anti-TH (1: 250, Pelfreeze), rabbit anti-Nurr1 (1: 1000, Santa Cruz), and rabbit anti-active caspase III (1: 100, Cell Signaling)) Was incubated overnight at 4 ° C.

  After washing 3 times with PBS, the sections were blocked with PBTG for 30 minutes and secondary antibodies (cyanine-2 or rhodamine-conjugated horse anti-mouse IgG (1: 200), or goat anti-rabbit IgG (1: 200, Jackson Laboratories For 2 hours. The slides were then washed 3 times with PBS for 15 minutes, counterstained with Hoechst 33258 for 1 minute, and then encapsulated in PBS / glycerin (1: 4). Images are captured at room temperature using a confocal laser scanning microscope (Zeiss, LSM510: argon (488 nm) and helium-neon (543 and 633 nm) lasers) and Zeiss LSM Viewer software, Adobe Photoshop version Image processed with 7.0. All panels were combined in Adobe Illustrator 9.0. Quantitative immunohistochemistry data are shown as mean ± standard error of the mean. All sections in which VM was present were counted in each animal, and 3-4 pairs of mice (wild type and LRP-6 mutant) were analyzed. Statistical analysis was performed by paired t-test using Prism 4 software (Graph Pad, San Diego, USA) as described. The significance of all tests was estimated at the level of p <0.05 (* p <0.05; ** 0.01 <p <0.001; *** p <0.001).

result
Continuous expression of Dkk1, Dkk2 and Dkk3 in the developing VM
The expression profile of Dkk was analyzed by real-time PCR in the developing rat VM. Dkk-1 was abundantly expressed at E10.5 to E11.5 and decreased at E13.5 (FIG. 1). However, Dkk-2 mRNA levels peaked at E11.5 (FIG. 2) (Perrone-Capano et al., 2000) before the initiation of DA neurogenesis in rats. Interestingly, this expression pattern is similar to that of Wnt-5a, a regulator that is important for DA differentiation previously reported (Castelo-Branco et al., 2003). Dkk-3 mRNA was also present at an early stage of VM development, but its expression was higher at the time after neurogenesis (E15.5 and P1, FIG. 3). Similar results were observed in the developing mouse VM by in situ hybridization. Dkk-1 was abundantly expressed in the midbrain curve (FIG. 4b), overlapping with the expression of TH (FIG. 4a). Furthermore, its expression was seen in a small area of the diencephalon. In E15.5, Dkk-1 expression was not observed in the midbrain (FIG. 4f). In E11.5, Dkk-2 was expressed in the diencephalon and midbrain curved part from the ventricle to the marginal zone, covering the TH expression region (FIG. 4c). Similar to that of Dkk-1, Dkk-2 mRNA was not found in the midbrain of E15.5 mice (FIG. 4g). The present inventors also examined whether postnatal VM glia (Wagner et al., 1999; Castelo-Branco et al., 2005), which is a DA-inducible signal source, express Dkk. Dkk-1 was not expressed in any of the analyzed glial cell groups, whereas Dkk-2 and Dkk-3 were found to be expressed in P1 VM glia (FIGS. 5 and 6). Interestingly, Dkk-2 expression was specific for VM glia, but unlike Dkk-3, it was not detected in cortical and striatum-derived glia (FIGS. 5 and 6). The dynamic pattern of Dkk expression in induced VM glia in vivo and after birth suggests distinct and non-overlapping functions of different Dkks in the developing VM.

  In order to investigate the effects of Dkk on developing VMs, we transiently transfected human embryonic kidney cells 293 (HEK293) with alkaline phosphatase (AP) -tagged Dkk (Mao et al., 2001a). . The conditioned medium was collected and partially purified, and the presence of Dkk-AP was confirmed by immunoblotting against AP and Dkk-3. Subsequently, the inventors treated E14.5 rat VM neural progenitor cell cultures with increasing doses of partially purified Dkk-AP and examined their effects on DA progenitor cell differentiation. Dkk-1 did not affect the number of tyrosine hydroxylase (TH) positive cells at any concentration of culture, whereas Dkk-2 significantly increased the number of DA neurons at 2.5 and 5 μl / ml (Fig. 7). Dkk did not affect the total number of Nurr1-positive cells in culture at any given dose (FIG. 8).

Partially purified Dkk conditioned medium and recombinant Dkk have the opposite effect on the differentiation of VM progenitor cells. Interestingly, when evaluating the number of DA progenitor cells (Nurr1 + / TH-) that turn to Nurr1 + / TH + DA neurons Dkk-2 showed a strong and lasting effect at 2.5-10 μl / ml, and Dkk-3 significantly increased its cell population at 2.5 μl / ml (FIGS. 9, 10). Thus, Dkk-2 appeared to be a strong inducer for VM DA neuron differentiation.

  In order to confirm these results, the present inventors used VM progenitor cell cultures with increasing doses of full-length recombinant mouse Dkk-1 (R & D Systems) and full-length recombinant human Dkk-2 (Aprox). ). As a result, the present inventors found that 5 to 10 ng / ml of Dkk-1 significantly reduced the number of TH + cells (FIG. 11). However, at higher doses (30-100 ng / ml), recombinant Dkk-1 no longer had an effect of reducing the number of TH + neurons (FIGS. 11, 12). Surprisingly, recombinant Dkk-2 was also found to reduce the number of TH + in VM progenitor cell cultures, although at higher concentrations (50-100 ng / ml) than recombinant Dkk-1. 13 and 14). Therefore, full-length recombinant Dkk has a different effect on DA neurogenesis compared to Dkk produced in mammalian cells. In summary, this data indicates that Dkk differentially regulates the differentiation of E14.5DA VM progenitor cells, Dkk-2 is a potent inducer of VM progenitor cell differentiation into TH + neurons, and Dkk- 1 indicates that it is an inhibitor.

Dkk is a full-length recombinant Dkk that undergoes proteolytic processing in developing VM-derived cells, and is produced in Escherichia coli. Therefore, it does not undergo post-translational modifications including proteolysis and glycosylation via precursor protein convertases similar to those occurring in mammalian cells. We therefore investigated whether such differences in processing were responsible for the difference in activity between full length Dkk and partially purified (pp) conditioned media (cm). Immunoblotting of ppcm with an antibody against AP revealed that both Dkk-1 and Dkk-2 exist in two forms. Apart from the high molecular weight band (approx. 105-110 kDa) corresponding to the AP tag (approx. 67 kDa) fusion full-length protein (approx. 35-40 kDa), an additional low molecular weight band (approx. Detected at 1 and Dkk-2 cm.

  Mouse and human Dkk-1 and Dkk-2 are potential minimal furin cleavage sites (Arg-XX-Arg), acidic pH furin cleavage sites (Arg / Lys-XXX-Arg / Lys-Arg), or A protease converting enzyme 2 (PC2) cleavage site (Arg / Lys-Arg) is contained in the center of the first cysteine-rich domain (CRD-1) (Rockwell et al., 2002; Thomas, 2002) (FIGS. 15 and 16). . In addition, Dkk-1 has a potential PC2 cleavage site in the spacer region between CRD-1 and the second rich domain (CRD-2), and also in CRD-2. Furthermore, it was found that furin and other precursor protein convertases are expressed in abundance during DA neuron formation in developing VMs (Zheng et al., 1994). This suggests that Dkk can be cleaved by proteolysis at this stage.

  To verify that Dkk is proteolytically processed, FLAG tag fusions Dkk-1 (about 40 kDa) and Dkk-2 (about 35 kDa) (Li et al., 2002) were overexpressed in HEK293T cells. The cells were treated with decanoyl RVKR-CMK (which is an inhibitor of proprotein convertases including furin and PC2) with increasing doses (Jean et al., 1998). 25 μM of the inhibitor was found to be sufficient to increase the total amount of uncleaved full length Dkk-1 or Dkk-2 fraction (approximately 35-40 kDa). These data indicate that the small molecular band of p.p.c.m. is indeed due to proteolytic processing. Furthermore, the inventors observed that this phenomenon also occurred in the SN4741 DA cell line (Son et al., 1999). When overexpressing FLAG-tagged Dkk, Dkk-1 is present in about 40 kDa full-length and about 25 kDa truncated forms, and treatment with decanoyl RVKR-CMK increases the total amount of full-length protein. (FIG. 17). It was also found that expression of Dkk-2 in SN4741 cells resulted in two truncated forms of approximately 22-25 kDa, but no full-length protein. These results suggest that Dkk undergoes different proteolytic cleavages in different mammalian cells including DA cells. Therefore, the present inventors next examined whether a cleaved fragment containing the complete cysteine-rich domain of Dkk-1 or Dkk-2 has a different biological activity.

Dkk-2 second cysteine-rich domain increases the number of TH-positive cells in VM progenitor cell cultures
Dkk-1 and Dkk-2 contain two cysteine-rich domains (CRDs) (Krupnik et al., 1999), and truncated proteins containing different CRDs differentially regulate canonical Wnt signaling activation. (Brott and Sokol, 2002; Li et al., 2002). However, since the furin and PC2 cleavage sites are within both Dkk-1 and Dkk-2 CRD-1 (FIGS. 15 and 16) and within Dkk-1 CRD-2, Dkk-2-CRD-2 Are likely to be simply truncated forms that mimic normal cleavage products. In order to investigate the activity of different CRDs of Dkk-1 and Dkk-2 in the development of VM DA neurons, we used D474-1 and Dkk-2 derived FLAG-tagged CRD (Li et al., 2002) to SN4741 A conditioned medium that was overexpressed in cells and partially purified was prepared. When different truncated proteins were expressed at moderate levels, they were present at the expected molecular weight (Li et al., 2002). Treatment of both Dkk-1 and Dkk-2 with CRD-1 increased the number of TH + cells at 2.5 μl / ml (FIGS. 18 and 19). Dkk-1 CRD-2 did not show a significant change in the number of DA neurons, but Dkk-2 CRD-2 increased at a dose of 2.5 μl / ml (FIG. 20). These results suggest that full length Dkk-1 and full length Dkk-2 have different activities in DA differentiation compared to their proteolytic fragments or CRD.

Full-length Dkk inhibits canonical Wnt signaling, whereas Dkk-2-CRD-2 has the opposite effect We investigated how Dkk regulates Wnt signaling in DA cell lines It was. Full-length recombinant Dkk-1 and full-length recombinant Dkk-2 canonical Wnt signaling, as shown by decreased levels of transcriptionally active β-catenin in SN4741 cells stimulated with low doses of Wnt-3a. Inhibited efficiently (FIG. 21). Interestingly, consistent with the effect of Dkk on VM progenitor cells, the effect of Dkk is less pronounced at higher doses of Wnt-3a, and Dkk-1 is β-catenin at lower doses than Dkk-2 Signal transduction could be inhibited. On the other hand, treatment of SN4741 cells with Dkk2-CRD-2 or Dkk-2 overexpressed and processed in SN4741 cells resulted in a synergistic effect on β-catenin activation via Wnt-3a. However, Dkk-1 or Dkk-1-CRD-2 had no effect. These results are due to Dkk-2 or Dkk-2-CRD-2 in the LRP-6 overexpressing cell line (Li et al., 2002; Liu et al., 2005), or Xenopus (Brott and Sokol, 2002). Consistent with previous reports showing activation of canonical Wnt signaling. Thus, our results indicate that the different activities of Dkk CDR and full-length protein control canonical Wnt signaling and DA neurogenesis, and proteolysis plays an important role in determining Dkk function. Suggests to fulfill.

LRP-5, LRP-6 and Kremen-1 are expressed in the developing VM, but Kremen-2 is not expressed, and LRP-6N-Fc promotes DA differentiation
Dkk-1 and Dkk-2 were originally identified as extracellular inhibitors of Wnt / β-catenin signaling (Kawano and Kypta, 2003). The inventors have also previously shown that glycogen synthase kinase (GSK) -3 inhibition / β-catenin stabilization leads to DA neuronal differentiation (Castelo-Branco et al., 2004). In order to control Wnt / β-catenin signaling, Dkk must bind to LRP-5 or LRP-6 (Bafico et al., 2001; Mao et al., 2001a; Semenov et al., 2001). Therefore, the present inventors analyzed the mRNA expression profile of LRP during VM development. Both LRP-5 and LRP-6 were expressed in VM (FIGS. 22, 23 and 24). Also interestingly, LRP-6 peaked at E11.5, as previously observed with Dkk-2 (Figure 2) and Wnt-5a (Castelo-Branco et al., 2003). High levels of LRP-6 can enhance canonical signaling through Wnt (Brennan et al., 2004; Holmen et al., 2002; Liu et al. 2003) and can gradually increase the inhibitory effect of Dkk-1 (Bafico et al., 2001; Mao et al. 2002; Mao et al., 2001a) and, along with high levels of Dkk-2, Wnt / β-catenin signaling in Xenopus (Brott and Sokol, 2002; Mao and Niehrs, 2003) and mammalian cells (Li et al., 2002). It has become clear that both Mao and Niehrs, 2003) can be activated. This suggests that Dkk-2 regulates DA differentiation through β-catenin activation. This process is also regulated by Kremen, a second class of Dkk co-receptors. Kremen-1 and Kremen-2 proteins can bind to Dkk and form a triple complex with LRP-6. This complex is then taken up into the cell and prevents signal transduction by Wnt / β-catenin (Mao et al., 2002).
However, Dkk cannot inhibit Wnt / β-catenin signaling when endogenous Kremen is limited or LRP5 / 6 levels are high (Davidson et al., 2002; Mao and Niehrs, 2003). For example, the lack of Kremen-2 can convert Dkk-2 from an inhibitor of canonical Wnt signaling to an activator (not possible with Dkk-1) (Mao and Niehrs, 2003). The inventors have found that Kremen-1 is present in the developing VM and peaks at E11.5, a stage where both Dkk-1 and Dkk-2 are still present at high levels (Fig. 25). However, Kremen-2 was barely detectable with real-time PCR.

  These results are consistent with Kremen's full in situ hybridization profile (Davidson et al., 2002), which showed that Kremen-1 is expressed in the initial VM but not Kremen-2. Our results suggest that both Dkk-1 and Dkk-2 interact with Kremen-1 in the initial VM and can inhibit Wnt canonical signaling.

  However, the lack of Kremen-2 is probably driving Dkk-2 to activate canonical Wnt signaling in various cell types of DA niche. Therefore, the present inventors examined the effects of treating E14.5 VM progenitor cell cultures with soluble Kremen-1 and Kremen-2 (R & D systems). Although the number of TH + cells tended to decrease, no statistically significant change was observed in any of them (FIGS. 26 and 27). Kremen membrane binding is essential for binding Dkk in 293T cells and Xenopus and for its Wnt inhibitory function (Davidson et al. 2002; Mao et al., 2002). This requirement may also apply to a running VM. In short, LRP and Kremen may be expressed in a specific pattern in the developing VM and play a role in DA differentiation.

  To further investigate the relevance of LRP-6 in our system, we used the secreted fusion protein LRP-6N-Fc containing the extracellular domain of FRP-6 and the Fc domain of human IgG. (Tamai et al., 2000). LRP-6N-Fc was overexpressed in HEK293 cells and its presence in the conditioned medium was confirmed by immunoblotting against human IgG. E14.5 VM progenitor cell cultures were then treated with increasing doses of LRP-6N-Fc to convert TH + neurons (FIG. 28A) and the percentage of Nurr1 progenitor cells converted to the DA phenotype (FIG. 28C). ) Found a strong effect on DA differentiation. The total number of Nurr1 positive cells was not affected (FIG. 28B). This suggests that the effect is on differentiation and not on proliferation of Nurr1 + progenitor cells. LRP-6N-Fc has been reported to weaken β-catenin stabilization via Wnt1 (Brennan et al., 2004; Gonzalez-Sancho et al., 2004; Holmen et al., 2002), which also binds to Dkk Yes (Semenov et al., 2001). We found that this fusion protein mimics the DA effect of Dkk-2 (FIG. 2) or Wnt-5a (Castelo-Branco et al., 2003) on DA differentiation.

LRP-6 homozygous mutant mice do not show patterning, proliferation or cell death defects in VM
LRP-6 homozygous mutant mice have been reported to exhibit several developmental defects associated with deregulation of Wnt signaling, including brain (encephaly) and / or caudal midbrain defects (Pinson et al., 2000). These previous observations were confirmed by decreased dorsal expression of midbrain / hindbrain marker genes such as Engrailed (En-1) and fibroblast growth factor 8 (fgf-8). However, no difference in the expression of En1, fgf-8, Sonic hedgehog (Shh), or Wnt-5a was observed in VMs of E11.5-12. This suggests that VM is normally formed with the mutant (FIG. 29). Therefore, defects in the midbrain-hindbrain boundary in LRP-6 homozygous mutants are likely associated with defects in dorsal / caudal midbrain structures.

  Consistent with the above findings, immunostaining was performed using BrdU (FIG. 30) as an S phase marker at E10.5 and E11.5 (FIG. 30) and phosphohistone 3 (FIGS. 31 and 32) as a cell cycle marker at E11.5. When evaluated, no decrease in proliferation of VM progenitor cells at E11.5 was observed (Hendzel et al., 1997). Furthermore, when evaluated by immunostaining of active caspase-3, the number of cells undergoing apoptosis did not change with E13.5 wild-type or mutant VM. The expression and distribution of nestin, a neural stem / progenitor cell marker (FIGS. 33 and 34), and the mRNA level of aldehyde dehydrogenase 2 (AHD-2), a DA progenitor cell marker (FIG. 35) (Wallen et al., 1999), It did not change with the mutant VM. These results suggest that deletion of LRP-6 does not alter the normal patterning, proliferation, or cell survival of DA precursor cells in VM.

Delayed DA neurogenesis in LRP-6 homozygous mutant mice We investigated whether LRP-6 regulates the late phase of DA development in VM. A marked decrease in the number of TH + cells (50%, Figures 36 and 37) was observed around E11.5, the time when DA neurons were born, but a general neuronal population (β-tubulin III (TUJ) immune response) There was no clear change in the property (FIG. 36). When assessed by real-time RT-PCR (FIG. 38) and in situ hybridization (FIG. 39), TH mRNA was also significantly lower in homozygous mutant mice. In E13.5, the difference in the number of DA cells between the wild type and the mutant type was reduced to 25% (FIGS. 40 and 41). In addition, no difference was detected in the mRNA level by real-time RT-PCR (FIG. 42). TH levels at E17.5 were normal in the substantia nigra and ventral tegmental area, two major DA nuclear regions in mutant mice (Figure 43). Furthermore, DA neurons were distributed in the striatum of mutant mice. Interestingly, a clear recovery of the number of DA neurons was consistent with increased expression of LRP-5 in the midbrain of homozygous mutant mice as assessed by in situ hybridization (FIG. 44).

  We next examined whether the expression of transcription factors required for normal development of DA neurons was altered in LRP-6 homozygous mutant mice. First, we focused on Nurr-1 (Castillo et al., 1998; Le et al., 1999; Zetterstrom et al., 1997), which is essential in the late phase of VMDA development including differentiation (Joseph et al., 2003). As a result, it was found that the number of Nurr-1 + cells (both TH-progenitor cells and TH + DA neurons) was greatly reduced in the E11.5 mutant VM (FIGS. 45 and 46). A 40% reduction in Nurr-1 mRNA levels at this stage was also observed by real-time PT-PCR (FIG. 47). However, in E13.5, the number of Nurr-1 + cells did not decrease even with the mutant VM (FIG. 48). Furthermore, it is expressed at an early stage of DA differentiation (Smidt et al., 1997) and required for its maintenance (Hwang et al., 2003; Maxwell et al., 2005; Nunes et al., 2003; Smidt et al., 2004; van den Munckhof et al., 2003) The expression of the transcription factor Pitx-3 was also greatly reduced in both E11.5 and E12.5 when assessed by real-time RT-PCR and in situ hybridization, respectively (FIGS. 49 and 50). However, the expression of these two transcription factors was close to normal levels by E13.5 (FIG. 51). Thus, our results indicate that lack of LRP-6 results in delayed DA neurogenesis. This suggests that although LRP-6 is not absolutely required in VMs, it is required for proper initiation of DA neuron formation.

  Dkk is the programmed cell death of developing limbs (Grotewold and Ruther, 2002), proliferation of adult human mesenchymal stem cells (Gregory et al., 2003), invasion and migration of osteosarcoma cells Involvement in the regulation of several cellular processes, including inhibition of (Hoang et al., 2004) and final osteoblast differentiation and calcified matrix formation (Li et al., 2005). In the present specification, (1) Dkk-1 and Dkk-2 are very important regulators of DA differentiation, and (2) their biological activities are regulated by proteolysis via precursor protein convertase. It has been demonstrated for the first time that (3) the LRP-6 receptor is required for the proper initiation of DA neurogenesis in the VM.

  The inventors have discovered that full-length recombinant Dkk-1 and Dkk-2 produced by Escherichia coli reduce the number of TH + neurons in rat E14.5 VM progenitor cell cultures. . However, partially purified Dkk conditioned media produced in mammalian cells (Mao et al., 2001a) differentially regulated DA differentiation. That is, partially purified Dkk-1 had no effect on DA neuron formation, and partially purified Dkk-2 enhanced differentiation of Nurr-1 + progenitor cells into TH + DA neurons. These results indicate that when full-length Dkk-2 is proteolyzed in the presence of Wnt, the second CRD of Dkk-2 may be involved in the activation effect of Dkk-2 on canonical Wnt signaling and DA neuron formation Suggests.

  Furin and other precursor protein convertases include several proteins such as nerve growth factor, Notch, α-amyloid precursor protein, tumor necrosis factor (TNF) member, and transforming growth factor β (TGF-β) family Is involved in the proteolytic maturation of (Thomas, 2002). The BMP-4 precursor undergoes sequential cleavage at two furin sites, which makes mature BMP-4 more stable (not a target for degradation), more active, and allows long-term signaling (Cui et al., 2001; Degnin et al., 2004). Our experiments using decanoyl RVKR-CMK, a common precursor protein convertor inhibitor, show that cleavage of full-length Dkk by members of the precursor protein convertase family greatly regulates Dkk's ability, and canonical Wnt It suggests that it regulates signal transduction and consequently regulates biological processes including DA neurogenesis. Interestingly, proprotein convertase is abundantly expressed in VM during neurogenesis (Zheng et al., 1994). Our data also indicate that 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 progenitor cells in the early stages of VM. Cleavage via furin or PC2 at the beginning of DA neuron formation inactivates Dkk-1 in VM, and in the presence of Wnt a second CRD turns Dkk-2 into a canonical Wnt pathway and an inducer of DA differentiation May be transformed (Castelo-Branco et al., 2003). This hypothesis is based on previous reports in Xenopus that Dkk-2 can activate canonical Wnt signaling in the absence of Kremen-2 and in the presence of high levels of LPR-6 (Brott and Sokol, 2002; Mao and Niehrs, 2003). In addition, Dkk-2-CRD-2 acts in conjunction with LRP-6 to induce secondary axis of Xenopus and the expression of Wnt / β-catenin target genes (Brott and Sokol, 2002). TCF / LEF-mediated transcription can be activated in mammalian cells overexpressing (Li et al., 2002). The effect of Dkk-2-CRD-2 as an activator is consistent with the results that we previously observed that β-catenin stabilization induces DA differentiation (Castelo-Branco et al., 2004). . Furthermore, it was recently found that a covalently linked chimera containing Wnt-3a and Dkk-2-CRD-2 activates canonical signaling (Li et al., 2005). Our results also showed that Wnt-3a and Dkk-2-CRD-2 can actually cooperate to activate canonical signaling. This suggests that Wnt and truncated Dkk together can regulate various signaling pathways and thereby control DA neurogenesis.

  The positive effect of Dkks-CRD-1 on DA neuron formation was also surprising. Because Dkk's first CRD cannot regulate canonical Wnt signaling (Brott and Sokol, 2002; Li et al., 2002) and cannot interact with LRP-6 (Brott and Sokol, 2002; Mao and Niehrs, 2003) It is. Our results suggest that these proteins can be used to modulate DA neurogenesis and therefore promote DA differentiation of neural stem or progenitor cells for therapeutic purposes.

  We also analyzed DA neurogenesis in mouse mutant VMs for the LRP-6 receptor. Importantly, it was found that the onset of DA differentiation was delayed with a 50% decrease in DA neurons at E11.5 and a 25% decrease at E13.5. The expression levels of Nurr-1 and Pitx-3, two important transcription factors involved in DA differentiation, were also temporarily low. These results indicate that elimination of the Dkk receptor LRP-6 in the VM (which mimics excessive Dkk-1 signaling, or diminished Dkk-2 and canonical Wnt signaling) disrupts DA differentiation. Emphasized. On the other hand, cell death, proliferation or pattern formation reported to be regulated by Wnt (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) found no difference. Similar results have been reported in the anterior segment mesoderm of LRP-6 hypomorph mice, with no difference in cell death and proliferation (Kokubu et al., 2004). A clear selectivity of functions regulated by Wnt in LRP-6 homozygous mutant mice has also been observed in other brain regions. The development of the dorsal thalamus is severely affected by LRP-6 homozygous mutants (with removal of Shh and Wnt-5a expression) (Zhou et al., 2004a). It was also reported that LRP-6 homozygous mutant mice showed neural progenitor cell defects in the dentate gyrus, but other cell types in the developing hippocampus were not affected (Zhou et al., 2004b). Therefore, our results showing that lack of LRP-6 affects DA neurogenesis emphasizes that LRP-6 is a general regulator of cell type-specific neurogenesis. ing.

  Interestingly, LRP6-/-mice were found to recover almost completely the substantia nigra and ventral tegmental area by E17.5 after the first loss of DA neurogenesis. These results suggest the existence of a compensatory mechanism that can restore the number of DA neurons and the expression of DA markers at later stages. One possibility for such a compensation mechanism may be LRP-5. This is because LRP-5 receptor expression is upregulated in LRP6-/-mice.

  In summary, our results indicate that LRP5 / 6 controls the onset of DA neurogenesis. It was also found that post-transcriptional processing of LRP6 ligand differentially regulates Wnt / β-catenin signaling. Importantly, proteolytically processed Dkk2, Dkk3 and Dkk-2-CRD-2 have been identified as candidates to enhance differentiation of DA progenitor cells into neurons. Thus, these factors may help promote DA neurogenesis in the context of (stem) cell replacement methods for Parkinson's disease treatment.

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2 shows real-time PCR analysis showing that Dkk-1 is expressed at a high level in the early stage in the ventral midbrain. The real-time PCR analysis which shows that Dkk-2 has an expression peak around E11.5 (embryonic day 11.5) is shown. The real-time PCR analysis which shows that Dkk-3 has an expression peak in the stage after E11.5 is shown. Shown are sagittal sections of mouse embryos at E11.5 and E15.5 hybridized with TH (a, e), Dkk1 (b, f), and Dkk2 (c, g) mRNA probes. In E11.5, TH is expressed in DA neurons in the midbrain curvature (a). Dkk1 is also expressed in the same region (b) and diencephalon. Dkk2 is expressed in the ventral marginal zone of the midbrain curve (c). In E15.5, TH is expressed at a high level in VM, but the expression of Dkk1 and Dkk2 is greatly reduced. (Explanation of symbols in the figure) te: telencephalon, ov: optic vesicle, di: diencephalon, mes: midbrain, hb: hindbrain, cx: cortex, cp: cerebellar primordium, rmd: highest midbrain Real-time PCR shows that P1 VM glia (VM G) express more Dkk-2 (B) than P1 cortex-derived glia (Cx G) and striatum-derived glia (Str G) . Real-time PCR shows that P1 VM glia (VM G) express Dkk-3 (B) at higher levels than P1 cortex-derived glia (Cx G) and striatum-derived glia (Str G) ing. Dkk-1 is not expressed in any glia. When E14.5 VM progenitor cells were treated with partially purified conditioned media (ppcm) from HEK 293T cells overexpressing Dkk-2-AP for 3 days, TH + cell count Is significantly increased by immunocytochemistry. On the other hand, Dkk-1 did not have any significant effect. Statistical analysis was performed using one-way analysis of variance and Fisher's post hoc multiple comparison test. This shows that treatment of VM progenitor cells with various Dkk-APs does not affect the number of Nurr1 immunoreactive cells. Statistical analysis was performed using one-way analysis of variance and Fisher's post hoc multiple comparison test. Treatment of E14.5 VM progenitor cells with increasing concentrations of Dkk-2-AP for 3 days shows a significant increase in the percentage of Nurr1 progenitor cells that acquired the TH positive phenotype. On the other hand, Dkk-3-AP had a significant effect only at 2.5 μl / ml. Dkk-1-AP had no effect. Statistical analysis was performed using one-way analysis of variance and Fisher's post hoc multiple comparison test. Double immunocytochemistry shows that treatment of E14.5 VM progenitor cells with increasing concentrations of Dkk-2-AP for 3 days significantly increases the percentage of Nurr1 progenitor cells that acquired the TH positive phenotype It is shown in the evaluation by. Treatment of E14.5 VM progenitor cells with 5-10 ng / ml recombinant Dkk-1 for 3 days reduces the percentage of TH immunoreactive cells in the total cell population compared to control (untreated) (Evaluated by Hoechst33258 staining). Statistical analysis was performed using one-way analysis of variance and Dunnett's multiple comparison test. Treatment of E14.5 VM progenitor cells with 5-10 ng / ml recombinant Dkk-1 decreased the percentage of TH immunoreactive cells in the total cell population compared to control (0 ng / ml). Shown (evaluated by Hoechst 33258 staining). Treatment of E14.5 VM progenitor cells with 50-100 ng / ml recombinant Dkk-2 for 3 days reduces the percentage of TH immunoreactive cells in the total cell population compared to control (untreated) (Evaluated by Hoechst33258 staining). Statistical analysis was performed using one-way analysis of variance and Dunnet's multiple comparison test. Treatment of E14.5 VM progenitor cells with 50-100 ng / ml recombinant Dkk-2 for 3 days reduces the percentage of TH immunoreactive cells in the total cell population compared to control (untreated) (Evaluated by Hoechst33258 staining). The sequences of human and mouse Dkk-1 and the PC2 cleavage fragment are shown. Mouse and human Dkk-1 are potential minimal furin cleavage sites (Arg-XX-Arg), acidic pH furin cleavage sites (Arg / Lys-XXX-Arg / Lys-Arg), or protease conversion The enzyme 2 (PC2) cleavage site (Arg / Lys-Arg) is contained in the middle of the first cysteine-rich domain (CRD-1) (Rockwell et al., 2002; Thomas, 2002). In addition, Dkk1 also has a potential PC2 cleavage site in the spacer region between CRD-1 and the second rich domain (CRD-2), and in CRD-2. According to these putative cleavage sites, during proteolysis, Dkk-1 has four fragments (bottom panel) that inactivate both the first (CRD-1) and second (CRD-2) cysteine-rich domains. Can be cut into pieces. The sequences of human and mouse Dkk-2, as well as the PC2 cleavage fragment are shown. Mouse and human Dkk-2 have potential minimal furin cleavage sites (Arg-XX-Arg), acidic pH furin cleavage sites (Arg / Lys-XXX-Arg / Lys-Arg), or protease convertase 2 ( PC2) contains a cleavage site (Arg / Lys-Arg) in the middle of the first cysteine-rich domain (CRD-1) (Rockwell et al., 2002; Thomas, 2002). According to these putative cleavage sites, Dkk-2 can be cleaved into two fragments. One of them contains functional CRD-2 (bottom panel). We show that Dkk proteolysis controls Dkk's effects in DA neurogenesis. A shows that ppcm derived from HEK293T cells overexpressing Dkk-1-AP and Dkk-2-AP has a high molecular weight (total length: about 105-110 kDa) recognized by human AP antibody and a low molecular weight (arrow and arrowhead, about The evaluation by Western blotting shows that it contains two bands of 80-85 kDa). The AP tag has a molecular weight of about 67 kDa. An equal amount of protein was run. B is treated with decanoyl-RVKR-CMK, a general inhibitor of precursor protein convertase, for 1 day when HEK293T cells overexpressing FLAG-tagged Dkk are treated with a full-length secreted, ie proteolytically degraded. An increase in the fraction of Dkk-1 and Dkk-2 not shown is shown by evaluation by Western blotting of conditioned medium (cm) using an anti-FLAG antibody. C shows p.p.c.m. derived from SN4741 cells overexpressing FLAG tag fusion Dkk by evaluation by Western blotting using an anti-FLAG antibody. The cell lysate derived from full length Dkk-1-FLAG showed two bands corresponding to full length protein (about 40 kDa) and proteolytic fragment (about 25 kDa, arrowhead). The full-length Dkk-2-FLAG is clearly completely proteolyzed because only two bands smaller than 25 kDa were detected (arrows). We show that Dkk proteolysis controls Dkk's effects in DA neurogenesis. Treating E14.5 VM progenitor cells with 2.5 μl / ml ppcm containing Dkk-1-CRD-1 (Dkk-1-C1) or Dkk-2-CRD-1 (Dkk-2-C1) The percentage of TH immunoreactive cells in the total cell population increased compared to the control ppcm (assessed by Hoechst33258 staining). Scale bar is 50 μm. Statistical analysis was performed using one-way analysis of variance and Dunnet's multiple comparison test. 2.5 μl containing Dkk-1-CRD-1 (Dkk-1-C1), Dkk-2-CRD-1 (Dkk-2-C1), or Dkk-2-CRD-2 (Dkk-2-C2) It has been shown that treatment of E14.5 VM progenitors with ppcm / ml increases the percentage of TH immunoreactive cells in the total cell population compared to the control ppcm (assessed by Hoechst33258 staining). Statistical analysis was performed using one-way analysis of variance and Dunnet's multiple comparison test. We show that Dkk proteolysis controls Dkk's effects in DA neurogenesis. When E14.5 VM progenitor cells were treated with 2.5 μl / ml ppcm containing Dkk-2-CRD-2 (Dkk-2-C2), the percentage of TH immunoreactive cells in the total cell population was controlled. Increased compared to ppcm (evaluated by Hoechst33258 staining). Statistical analysis was performed using one-way analysis of variance and Dunnet's multiple comparison test. Recombinant Dkk-1 (A) or recombinant Dkk-2 (B) uses antibodies against active β-catenin to inhibit basal or Wnt-3a canonical Wnt signaling in SN4741 cells Western blot evaluation. (C) From the evaluation by Western blot using an antibody against active β-catenin, ppcm containing Dkk-2 or Dkk-2-CRD-2 (10 μl / ml) is co-treated with Wnt-3a in SN4741 cells Synergistic effects on basal canonical Wnt signaling. Shows real-time RT-PCR analysis showing that LRP-5 is expressed at moderate levels in the ventral midbrain. The real-time RT-PCR analysis which shows that LRP-6 has an expression peak around E11.5 is shown. The real-time RT-PCR analysis which shows that Kremen-1 has an expression peak around E11.5 is shown. Kremen-2 is not expressed. The figure shows a sagittal section of a mouse embryo in E11.5 hybridized with LRP-5 and LRP-6 mRNA probes for in situ hybridization. Both genes are ubiquitously expressed in all brain structures, including VM regions with TH + DA neurons. (Explanation of symbols in the figure) te: telencephalon, ov: optic vesicle, di: diencephalon, mes: midbrain, hb: hindbrain It shows that recombinant Kremen-1 does not affect the percentage of TH positive cells from VM progenitor cells at E14.5 compared to control (untreated). Statistical analysis was performed using one-way analysis of variance and Dunnet's multiple comparison test. The concentration unit is ng / ml. It shows that recombinant Kremen-2 does not affect the percentage of TH positive cells from VM progenitor cells at E14.5 compared to control (untreated). Statistical analysis was performed using one-way analysis of variance and Dunnet's multiple comparison test. The concentration unit is ng / ml. A to C show that LRP-6N-Fc significantly increases DA differentiation of E14.5 VM progenitor cells. A shows by immunocytochemistry that the number of tyrosine hydroxylase (TH) positive cells significantly increases when E14.5 VM progenitor cells are treated with LRP-6N-Fc at increasing concentrations for 3 days. Is shown. B shows that the number of Nurr1-positive cells is not affected by the treatment of VM progenitor cells with LRP-6N-Fc. C shows that treatment of VM progenitor cells with LRP-6N-Fc significantly increases the percentage of Nurr1 progenitor cells that acquired the TH positive phenotype. Statistical analysis was performed using one-way analysis of variance and Dunnet's multiple comparison test. It shows that expression of midbrain pattern forming genes such as En-1, Fgf-8 and Shh is not changed in VM of LRP-6 homozygous mutant mice at E12.5 compared to control. The immunohistochemical evaluation of E10.5 against BrdU shows that the proliferative ability of E11.5 VM progenitor cells is not affected in LRP-6 homozygous mutant mice. The evaluation by cell count using anti-phosphohistone 3 antibody indicates that the proliferative ability of E11.5 VM progenitor cells is not affected in LRP-6 homozygous mutant mice (n = 3). Statistical analysis was performed using a paired t-test. An evaluation by immunohistochemistry using anti-phosphohistone 3 antibody shows that the proliferative ability of E11.5 VM progenitor cells is not affected in LRP-6 homozygous mutant mice. Real-time RT-PCR of LRP-6 homozygous mutant VM using primers for nestin, a progenitor cell marker, that nestin expression in VM precursor cells of E11.5 is not affected in LRP-6 homozygous mutant mice This is shown in the evaluation by analysis (n = 4). Statistical analysis was performed using a paired t-test. Nestin expression in E11.5 VM progenitor cells is not affected by LRP-6 homozygous mutant mice, as shown by immunohistochemical evaluation using an antibody against the progenitor cell marker nestin. The expression of AHD-2 in E11.5 VM progenitor cells is not affected in LRP-6 homozygous mutant mice, using LRP-6 homozygous mutant VM using primers for AHD-2, a DA precursor cell marker. This is shown in the evaluation by real-time RT-PCR analysis. Statistical analysis was performed using a paired t-test. A coronal section through the E11.5 VM is shown. This figure revealed that the number of DA neurons was reduced in LRP-6 homozygous mutant mice (as assessed by TH immunostaining) compared to controls. This decrease occurred mainly in midline DA neurons. General neuronal differentiation (as assessed by β-tubulin III (TUJ)) was unaffected, but only nerve fibers were seen in the median marginal zone and no neuronal cell bodies were seen. Shown is the count of TH + cells (at all levels) in the E11.5 VM of LRP-6 mutant mice (as assessed by TH immunostaining). This shows a 50% decrease in LRP-6 mutant mice when compared to controls (n = 3). Neuronal differentiation (as assessed by β-tubulin III (TUJ)) appears to be unaffected. Statistical analysis was performed using a paired t-test. The count of total TH + cells (at all levels) in the E11.5 VM of LRP-6 mutant mice is shown by evaluation by real-time RT-PCR. The figure shows a similar decrease in TH mRNA levels in the VM of LRP-6 homozygous mutant mice (n = 6). Statistical analysis was performed using a paired t-test. In situ hybridization using a TH probe is shown. This figure shows a decrease in TH mRNA levels in VM of LRP-6 homozygous mutant mice at E11.5. A coronal section through the E13.5 VM is shown. From this figure, it became clear that the number of DA neurons in LRP-6 homozygous mutant mice partially recovered compared to E11.5 stage. Shown is the count of TH + cells (at all levels) in the E13.5 VM of LRP-6 mutant mice (as assessed by TH immunostaining). This showed a 25% decrease in LRP-6 mutant mice when compared to controls (n = 4). Statistical analysis was performed using a paired t-test. This shows that the TH mRNA level in VM was not significantly reduced at E13.5 in LRP-6 homozygous mutant mice (n = 3). Statistical analysis was performed using a paired t-test. Immunostaining showing that E17.5 DA neuron counts were fully restored in the substantia nigra (SN) and ventral tegmental area (VTA) nuclei of LRP-6 homozygous mutant VM Represents the result. The sagittal section evaluated by in situ hybridization is shown. This figure shows that LRP-5 mRNA was increased in the E15.5 LRP-6 homozygous mutant VM, but LRP-6 mRNA could not be detected. A coronal section through the E11.5 VM is shown. This figure revealed that the number of Nurr1 + progenitor cells decreased in LRP-6 homozygous mutant mice compared to controls. It shows that the total number of Nurr1 + cells in E11.5 VM (in all horizontal planes) was reduced in LRP-6 homozygous mutant mice compared to controls (n = 3). Statistical analysis was performed using a paired t-test. A real-time PCR analysis of VM from LRP-6 homozygous mutant mice revealed a similar decrease in Nurr1 mRNA levels (n = 6). Statistical analysis was performed using a paired t-test. Immunostaining for Nurr1 in a sagittal section through the VM of E13.5, revealing that the number of Nurr1 + progenitor cells at E13.5 in LRP-6 homozygous mutant mice was not reduced compared to controls Real time RT-PCR is shown (n = 4). Real-time RT-PCR analysis of VM from E11.5 LRP-6 homozygous mutant mice showed a decrease in Pitx-3 mRNA levels (n = 5). Statistical analysis was performed using a paired t-test. In situ hybridization shows that the expression of Pitx-3, a marker of DA differentiation, decreased in E12.5 VM. E13.5 LRP-6 homozygous mutant mouse VM shows no decrease in the number of Nurr1 + progenitor cells at this stage compared to controls (n = 4). Statistical analysis was performed using a paired t-test.

Claims (114)

Induces the development of dopaminergic neurons by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and / or maturation in neural stem cells, embryonic stem cells, progenitor cells or progenitor cells A way to promote,
Modulate the level and / or activity of a Dkk ligand / receptor polypeptide in the cell;
Thereby inducing or enhancing proliferation, self-renewal, survival, and / or induction of dopaminergic, differentiation, survival or acquisition of a neuronal dopaminergic phenotype,
Said method comprising.   2. The method of claim 1, comprising treating the cell with a Dkk ligand / receptor polypeptide.   3. The method of claim 2, wherein the Dkk ligand / receptor polypeptide is a Dkk1, Dkk2, Dkk-3 or Dkk4 polypeptide.   4. The method of claim 3, wherein the polypeptide comprises cysteine rich domain 1 and / or cysteine rich domain 2 from human Dkk1, Dkk2, Dkk3 or Dkk4.   5. The method of claim 3 or 4, wherein the Dkk polypeptide is a Dkk1 polypeptide having at least 50% sequence identity to human Dkk-1.   5. The method of claim 3 or 4, wherein the Dkk-2 polypeptide has at least 50% sequence identity to human Dkk-2.   5. The method of claim 3 or 4, wherein the Dkk-3 polypeptide has at least 50% sequence identity to human Dkk-3.   5. The method according to claim 3 or 4, wherein the Dkk-4 polypeptide has at least 50% sequence identity to human Dkk-4.   3. The method of claim 1 or 2, wherein the Dkk ligand / receptor polypeptide is an LRP polypeptide.   10. The method of claim 9, 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.   11. The method of claim 10, wherein the LRP polypeptide is an LRP-5 polypeptide comprising the extracellular domain of human LRP-5.   12. The method of claim 10 or 11, wherein the LRP polypeptide comprises an amino acid sequence having at least 50% sequence identity to human LRP-5.   13. The method of claim 12, wherein the LRP polypeptide comprises the amino acid sequence of human LRP-5.   10. The method of claim 9, 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.   15. The method of claim 14, wherein the LRP polypeptide comprises the extracellular domain of human LRP-6.   16. The method of claim 14 or 15, wherein the LRP polypeptide comprises an amino acid sequence having at least 50% sequence identity to human LRP-6.   17. The method of 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.   8. The method of claim 7, wherein the Dkk ligand / receptor polypeptide further comprises an immunoglobulin Fc domain.   20. The method of any one of claims 2-19, wherein a Dkk ligand / receptor polypeptide is added to an in vitro culture containing the cells.   A Dkk ligand / receptor polypeptide is produced by expression from a cell co-cultured with said stem cell, embryonic stem cell, neural stem cell, progenitor cell or progenitor cell, and the co-cultured cell is type 1 astrocyte or early A cell other than a glial cell, or a host cell transformed with a nucleic acid encoding a Dkk ligand / receptor polypeptide or a cell containing an introduced Dkk ligand / receptor polypeptide. The method of any one of these.   The method according to claim 21, wherein the co-cultured cells or host cells other than type 1 astrocytes or early glial cells are other stem cells, embryonic stem cells, neural stem cells, progenitor cells, progenitor cells or neurons.   23. The stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neural cell is genetically engineered to express a Dkk ligand / receptor polypeptide from the encoding nucleic acid. The method described in 1.   24. The method of claim 22, wherein the Dkk ligand / receptor polypeptide is introduced into the cells to be co-cultured.   2. The method of claim 1, comprising treating the cell with a non-peptidic Dkk ligand.   2. The method of claim 1, comprising treating the cell with a compound that is an inhibitor, blocker, antagonist or suppressor of a Dkk ligand / receptor polypeptide.   27. The method of claim 26, wherein the compound is a Dkk1 or Dkk4 inhibitor, blocker, antagonist or suppressor.   2. The method of claim 1, wherein the level of Dkk ligand / receptor polypeptide is modulated by modulating the activity of a precursor protein convertase in the cell.   29. The method of claim 28, wherein the precursor protein convertase specifically cleaves the sequence Arg-X-X-Arg, Arg / Lys-X-X-X-Arg / Lys-Arg, or Arg / Lys-Arg.   30. The method of claim 29, wherein the precursor protein convertase is furin or protease convertase 2 (PC2).   31. The method of any one of claims 1-30, wherein the cell expresses a Nurr1 subfamily nuclear receptor.   32. The method of claim 31, wherein the nuclear receptor is Nurr1.   33. The any one of claims 1-32, wherein the stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neural cell is mitotic and / or capable of self-renewal. the method of.   2. The stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neural cell is further contacted with a member of a ligand of the Wnt family or a Wnt upstream regulator such as Msx1. 34. The method according to any one of -33.   The stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or nerve cell is retinoid or retinoid derivative, retinoid X receptor (RXR) activator, retinoic acid receptor (RAR) 35. The method of any one of claims 1-34, wherein the method is contacted with an inhibitor, 9-cis retinal, DHA, SR11237, or LG849.   Said stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neuronal cell prior to or modulating the level and / or activity of a Dkk ligand / receptor polypeptide in said cell 36. The method according to any one of claims 1 to 35, wherein the treatment is performed simultaneously with a member of a growth factor of the FGF family.   Treat the stem cells, embryonic stem cells, neural stem cells, progenitor cells, progenitor cells, or other stem cells or neurons with bFGF and / or EGF and / or FGF-8 and / or LIF and / or Shh and / or insulin 40. The method of claim 36.   Stem cells, embryonic stem cells, neural stem cells, progenitor cells, progenitor cells, or other stem cells or neurons, antioxidants, ascorbic acid, hypoxic partial pressure, hypoxia inducing factor, neurotrophic factor, GDNF, engrailed 38. The method of any one of claims 1-37, wherein the method is grown in the presence of, Pax2, Mash1, neurogenin, Pitx3 or BclxL.   The stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neural cell grows and / or differentiates in the presence of ventral midbrain astrocytes or early glial cells. 40. The method according to any one of 38.   Further co-culturing said stem cells, embryonic stem cells, neural stem cells, progenitor cells, progenitor cells, or other stem cells or neurons with primary glial cells or (optionally ventral mesencephalic) type 1 astrocytes 40. The method of any one of claims 1-39.   41. The method of claim 40, wherein the type 1 astrocytes are immortalized or derived from a cell line of an astrocyte in a region other than the ventral midbrain.   The method further comprising contacting the stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neuron with a negative selection agent that selects for non-dopaminergic neurons. 42. The method according to any one of 41.   Subjecting said neural stem cell, embryonic stem cell, progenitor cell or progenitor cell to oxidative stress prior to modulating the level and / or activity of a Dkk ligand / receptor polypeptide in said cell. 43. The method according to any one of 42.   44. The method of any one of claims 1-43, further comprising formulating the treated neurons into a composition containing one or more additional components.   45. The method of claim 44, wherein the composition contains a pharmaceutically acceptable additive.   46. The method of claim 45, further comprising administering the composition to an individual.   48. The method of claim 46, wherein the neurons are transplanted into the individual's brain.   20.The stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neuronal cell is treated with a Dkk ligand / receptor polypeptide at its site of development in an individual. The method according to any one of the above.   49. The method of claim 48, wherein a nucleic acid encoding a Dkk ligand / receptor polypeptide is introduced into the cell.   49. The method of claim 48, wherein a Dkk ligand / receptor polypeptide is introduced into the cell.   51. The method of any one of claims 48-50, wherein the stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neuronal cell is endogenous to the individual.   51. The method of any one of claims 48-50, wherein the stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neuronal cell is supplied exogenously by transplantation into the individual. The method described.   53. The method of any one of claims 48 to 52, wherein the individual has Parkinson's disease, Parkinson's syndrome, neuronal loss, or neurodegenerative disease.   44. The method of any one of claims 1-43, further comprising using neurons obtained by the method in the manufacture of a medicament for treating an individual.   55. The method of claim 54, wherein the medicament is for transplantation into an individual's brain.   56. The method of claim 55, wherein the individual has Parkinson's disease, Parkinsonism, neuronal loss, or neurodegenerative disease.   44. A dopaminergic neuron obtained by the method according to any one of claims 1 to 43.   58. Use of a dopaminergic neuron according to claim 57 in a method for screening a drug for treating a neurodegenerative disease. A method for screening a drug for treating a neurodegenerative disease,
(i) treating dopaminergic neurons obtained by the method according to any one of claims 1 to 43 with a toxin of the dopaminergic neurons;
(ii) separating the dopaminergic neurons from the toxin;
(iii) contacting the dopaminergic neurons with one or more test agents;
(iv) measuring the ability of the dopaminergic neurons to recover from the toxin;
(v) comparing the ability of the dopaminergic neurons to recover from the toxin with the ability of dopaminergic neurons not to contact the test agent to recover from the toxin;
Said method comprising. A method for screening a drug for treating a neurodegenerative disease,
(i) treating the dopaminergic neuron according to claim 57 with a toxin of the dopaminergic neuron in the presence of one or more test agents;
(ii) measuring the resistance of the dopaminergic neurons to the toxin;
(iii) comparing the resistance of the dopaminergic neurons to the toxin with that of dopaminergic neurons not contacted with the test agent;
Said method comprising. A method for screening a drug for treating a neurodegenerative disease,
(i) contacting dopaminergic neurons obtained by the method of any one of claims 1 to 43 with one or more test agents;
(ii) confirming the growth or viability of the dopaminergic neurons or the release of neurotransmitters in the presence of the agent;
Said method comprising.   62. The method of claim 61, wherein the dopaminergic neuron arises from a stem cell, progenitor cell or progenitor cell that has been exposed to oxidative stress.   63. The method of any one of claims 60-62, further comprising formulating the one or more test agents into a composition containing one or more additional ingredients.   64. The method of claim 63, wherein the composition contains a pharmaceutically acceptable additive.   65. The method of claim 64, further comprising administering the composition to an individual.   66. The method of claim 65, wherein the individual has Parkinson's disease, Parkinsonism, neuronal loss, or neurodegenerative disease. One or more that enhances proliferation, self-renewal, survival, and / or dopaminergic neurotransmission, development, induction, differentiation or maturation alone or in combination in stem cells, embryonic stem cells, neural stem cells, progenitor cells, or progenitor cells A method for obtaining the factor of
(a) treating neural stem progenitor or progenitor cells with Dkk ligand or Dkk receptor in the presence or absence of one or more test substances;
(b) measuring the proliferation, self-renewal, survival, and / or dopaminergic neurotransmission, development, induction, differentiation or maturation of the cells, and the proliferation in the presence or absence of the test substance, Compare the degree of self-renewal, survival, and / or dopaminergic neurotransmission, development, induction, differentiation or maturity, thereby obtaining the factor;
Said method comprising. One or more that enhances proliferation, self-renewal, survival, and / or dopaminergic neurotransmission, development, induction, differentiation or maturation alone or in combination in stem cells, embryonic stem cells, neural stem cells, progenitor cells, or progenitor cells A method for obtaining the factor of
(a) treating neural stem progenitor or progenitor cells with Dkk ligand or Dkk receptor in the presence or absence of one or more test substances;
(b) measuring β-catenin activation in the cells and comparing the degree of β-catenin activation in the presence or absence of the test substance, thereby obtaining the factor;
Said method comprising.   69. The method of claim 67 or 68, wherein the neural stem progenitor or progenitor cell is treated with a Dkk ligand / receptor polypeptide.   70. The method of claim 69, wherein the Dkk ligand / receptor polypeptide is a Dkk1, Dkk-2, Dkk3 or Dkk4 polypeptide.   71. The method of claim 69 or 70, wherein the Dkk ligand / receptor polypeptide comprises cysteine rich domain 1 and / or cysteine rich domain 2 from human Dkk1, Dkk2, Dkk3 or Dkk4.   72. The method of claim 70 or 71, wherein the Dkk ligand / receptor polypeptide is a Dkk1 polypeptide having at least 50% sequence identity to human Dkk-1.   72. The method of 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.   72. The method of 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.   72. The method of 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.   70. The method of claim 69, wherein the Dkk ligand / receptor polypeptide is an LRP polypeptide.   77. The method of 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. The method of claim 77, wherein the LRP polypeptide is an LRP-5 polypeptide comprising the extracellular domain of human LRP-5.   78. The method of claim 77 or 78, wherein the LRP polypeptide comprises an amino acid sequence having at least 50% sequence identity to human LRP-5.   80. The method of claim 79, wherein the LRP polypeptide comprises the amino acid sequence of human LRP-5.   77. The method of 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.   84. The method of claim 81, wherein the LRP polypeptide is an LRP-6 polypeptide comprising the extracellular domain of human LRP-6.   84. The method of claim 81 or 82, wherein the LRP polypeptide comprises an amino acid sequence having at least 50% sequence identity to human LRP-6.   84. The method of claim 83, wherein the LRP polypeptide comprises the amino acid sequence of human LRP-6.   85. The method of any one of claims 76-84, wherein the LRP polypeptide further comprises one or more additional amino acids.   86. The method of claim 85, wherein the LRP polypeptide further comprises an immunoglobulin Fc domain.   87. The cell of any one of claims 68-86, wherein the cell is treated with the Dkk ligand / receptor polypeptide by adding a Dkk ligand / receptor polypeptide to an in vitro culture containing the cell. Method.   87. The method of any one of claims 68 to 86, wherein the cell is treated with the Dkk ligand / receptor polypeptide by introducing a nucleic acid encoding a Dkk ligand / receptor polypeptide into the cell.   87. The method of any one of claims 68 to 86, wherein the cell is treated with a Dkk ligand / receptor polypeptide by introducing a Dkk ligand / receptor polypeptide into the cell.   Co-culture with cells other than type 1 astrocytes or early glial cells, host cells transformed with nucleic acid encoding Dkk ligand / receptor polypeptide, or cells containing introduced Dkk ligand / receptor polypeptide 87. The method of any one of claims 68 to 86, wherein the stem cell, embryonic stem cell, neural stem cell, progenitor cell, progenitor cell, or other stem cell or neuronal cell is treated with a Dkk ligand / receptor polypeptide. the method of.   Further comprising co-culturing said stem cells, embryonic stem cells, neural stem cells, progenitor cells, progenitor cells, or other stem cells or neurons with primary glial cells or (optionally ventral mesencephalic) type 1 astrocytes 87. A method according to any one of claims 68 to 86. Induces the development of dopaminergic neurons by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and / or maturation in neural stem cells, embryonic stem cells, progenitor cells or progenitor cells A method of screening for compounds useful for promoting,
Confirming the ability of the proprotein convertase to cleave the Dkk polypeptide by proteolysis in the presence of a test compound;
In this case, the above-mentioned method wherein the ability of the test compound to increase or decrease in the presence compared to the absence of the test compound indicates that the test compound is useful for inducing or promoting the development of dopaminergic neurons.   93. The method of claim 92, wherein the ability of the precursor protein convertase to cleave the Dkk polypeptide by proteolysis is confirmed by measuring Dkk activity in the presence of the precursor protein convertase.   94. The method of claim 93, wherein Dkk activity is confirmed by measuring β-catenin activation or dopaminergic development.   94. The method of claim 92, wherein the ability of the proprotein convertase to cleave a Dkk polypeptide by proteolysis is determined by confirming the presence of a product of proteolytic cleavage of the Dkk polypeptide.   96. The 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.   96. The method of any one of claims 92 to 96, wherein the precursor protein convertase is specific for the sequence Arg-XX-Arg, Arg / Lys-XXX-Arg / Lys-Arg, or Arg / Lys-Arg. .   98. The method of claim 97, wherein the precursor protein convertase is furin or protease convertase 2 (PC2).   A single compound or factor that can enhance proliferation, self-renewal, survival, and / or dopaminergic neurotransmission, development, induction, differentiation or maturation in stem cells, embryonic stem cells, neural stem cells, progenitor cells, or progenitor cells 99. A method according to any one of claims 68 to 98, provided in isolated and / or purified form.   A compound or factor capable of enhancing proliferation, self-renewal, survival and / or dopaminergic neurotransmission, development, induction, differentiation or maturation in a stem cell, embryonic stem cell, neural stem cell, progenitor cell or progenitor cell, 100. The method of any one of claims 68-99, formulated into a composition containing one or more additional ingredients.   101. The method of claim 100, wherein the composition comprises stem cells, embryonic stem cells, neural stem cells, progenitor cells, or progenitor cells.   102. The method of claim 100 or 101, wherein the composition comprises a DKK ligand / receptor polypeptide.   The method according to any one of claims 100 to 102, wherein the composition comprises a pharmaceutically acceptable additive.   104. The method of claim 103, further comprising administering the composition to an individual.   105. The method of claim 104, wherein the composition is transplanted into an individual's brain.   106. The method of claim 105, wherein the individual has Parkinson's disease, Parkinson's syndrome, neuronal loss, or a neurodegenerative disease. A method for screening a Dkk ligand that is not prone to protein hydrolysis,
Confirming the Dkk activity of a sample expected to contain a Dkk ligand in the presence of a precursor protein convertase,
The above method, wherein the presence of activity indicates that the sample contains the Dkk ligand.   The Dkk activity of the sample increases dopaminergic activity by enhancing proliferation, self-renewal, dopaminergic neurotransmission, development, induction, survival, differentiation and / or maturation in neural stem cells, embryonic stem cells, progenitor cells, or progenitor cells 108. The method of claim 107, wherein the method is confirmed by measuring the effect of the sample on promoting the development of sex neurons.   108. The method of claim 107, wherein the Dkk activity of the sample is confirmed by measuring β-catenin activation.   108. The method of claim 107, wherein the Dkk activity of the sample is confirmed by confirming the presence of a product of proteolytic cleavage of a Dkk ligand.   111. The method according to any one of claims 107 to 110, wherein the precursor protein convertase is specific for the sequence Arg-XX-Arg, Arg / Lys-XXX-Arg / Lys-Arg, or Arg / Lys-Arg. .   112. The method of claim 111, wherein the precursor protein convertase is furin or protease convertase 2 (PC2).   113. The method of any one of claims 107 to 112, comprising identifying the sample as containing a Dkk ligand that is not prone to proteolysis.   114. The method of claim 113, comprising isolating and / or purifying Dkk ligand from the sample.

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