Methods of Increasing Neurogenesis

This invention relates to the promotion of neurogenesis, in particular dopaminergic neurogenesis in progenitor or precursor cells.

Most neurodegenerative diseases affect neuronal populations . Moreover, most of the damage occurs to cells with a specific neurochemical phenotype. In human Parkinson's disease, for example, the major cell type lost is midbrain dopaminergic neurons.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects 1% of the population aged over 50 years. It is- characterized by a slowness, and difficulty in initiating movements (Arenas, 2005; Maxwell and Li, 2005; Deuschl et al . , 2006). The hallmark pathologic feature of PD is loss of melanized dopaminergic neurons (DNs) within the substantia nigra pars compacta, coupled with depletion of striatal dopamine, which is responsible for the major motor features of the disease (Snyder and Olanow, 2005) . Pharmacological dopaminergic replacement therapy with L-DOPA is effective in the early stages of the illness, but chronic treatment is associated with motor complications.

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 0., Pharmacol Res. 47 (4) -.279-87 (2003); Bjδrklund A. et al . , Lancet Neurol . 2(7):437-45 (2003), Snyder and Olanow, 2005; Winkler et al., 2005.

Intrastriatal transplants of human foetal mesencephalic tissue in Parkinson's patients have demonstrated clinical efficacy (Arenas, 2005, Lindvall et al., 2004), but ethical issues and the limited

availability of tissue, have precluded the systematic use of this treatment (Arenas, 2005; Taylor and Minger, 2005) .

Stem/progenitor or precursor cells are an ideal material for transplantation therapy since they can be expanded and instructed to assume specific neuronal phenotypes which fulfil the specific criteria for cell-replacement therapies. These cells would circumvent ethical and practical issues surrounding the use of human foetal tissue for transplantation. In particular, implanted non- autologous tissue has a limited viability and may be rejected by the immune system. In addition, each foetus provides only a small number of cells.

Stem, cells, including embryonic stem cells (ESs) and adult stem cells, are characterized by their extensive self-renewal capacity, and their potential to differentiate into any cell type of the body. In particular, stem cells have the ability to differentiate into neural cell lineages including neurons, astrocytes and oligodendrocytes. Moreover, stem cells can be isolated, expanded, and used as source material for brain transplants (Snyder, E. Y. et al. Cell 68, 33-51 (1992); Rosenthal, A. Neuron 20, 169-172 (1998); Bain et al., 1995; Gage, F. H. et al. Ann. Rev. Neurosci. 18, 159-192 (1995); Okabe et al . , 1996; Weiss, S. et al. Trends Neurosci. 19, 387-393 (1996); Snyder, E. Y. et al . Clin. Neurosci. 3, 310-316 (1996); Martinez-Serrano, A. et al . Trends Neurosci. 20, 530-538 (1997); McKay, R. Science 276, 66-71 (1997); Deacon et al., 1998; Studer, L. et al . Nature Neurosci. 1, 290-295 (1998); Bjorklund and Lindvall 2000; Brustle et al., 1999; Lee et al., 2000; Shuldiner et al., 2000 and 2001; Reubinoff et al., 2000 and 2001; Tropepe et al . , 2001; Zhang et al . , 2001; Price and Williams 2001; Arenas 2002;

Bjorklund et al., 2002; Rossi and Cattaneo, 2002; Gottlieb et al . , 2002) .

Midbrain dopaminergic neurons have been induced from mice/human ESCs by different methods (Andersson et al . , 2006; Roy et al . , 2006; Yan et al., 2005; Perrier et al . , 2004; Barberi et al . , 2003; Kim et al., 2002; Lee et al . , 2000; Kawasaki et al . , 2000:). In a recent study, Roy et al . , 2006, provided the first evidence of in vivo survival of dopaminergic neurons derived from hESs after transplantation in a rat model of PD. However, the grafts exhibited phenotype instability, and persistent proliferation of undifferentiated cells in the transplant cores, suggesting that the cells are not properly differentiated and their properties are not identical to fetal midbrain DNs.

In order to achieve an efficient DN differentiation of stem cells, appropriate milieu in the ventral midbrain must be provided for their development and this milieu is provided in great part by radial glial (RG) cells with which they are in direct contact from embryonic development to adulthood.

The development of the ventral midbrain results from a concerted action by secreted factors emanating from the floorplate and the midbrain-hindbrain organizer (MHO) and transcription factors expressed in the midbrain regulate the expression of key transcriptional networks controlling several aspects of the development the ventral midbrain including neurogenesis and DA neuron development.

A first important pathway is involves the regulation of Wntl by several factors including the homeodomain transcription factor Otx2 (Puelles et al . , 2004), a factor emanating from the hindbrain side of the MHO, FGF8 (Prakash et al., 2006), and the homeodomain transcription factor, Lmxlb (Matsunaga et al., 2002). Interestingly, deletion of any of theses genes, otxl/2, wntl or lmxlb, results in the loss of DA neurons (Puelles et al . , 2004; Prakash et al., 2006; Anderson et al 2006) . In its turn, the function of Wntl is to

regulate several aspects of midbrain development (McMahon and Bradley, 1990; Thomas and Capecchi, 1990), including the creation of an nkx2.2 free domain in the floor plate, the proliferation of DA progenitors, and the expression of pitx3 (Danieleian and McMahon, 1996; Prakash et al . , 2006. Castelo-Branco et al . , 2003), a homeobox required for DA neuron survival, that is also regulated by Lmxlb (Smidt et al.2000) .

A second pathway involves the expression of Shh, which is expressed in the floor plate and involved in ventral patterning that triggers the expression of two downstream transcription factors, Lmxla and Msxl, which are involved in the specification of the DA phenotype and the acquisition of a pan-neuronal phenotype, respectively (Andersson et al . , 2006).

The liver X receptors (LXRs) constitute a subfamily within the nuclear receptor superfamily of transcription factors. Two different members have been identified, LXR α and β that form obligate heterodimers with retinoid X receptor (RXR) (Edwards et al . , 2002). LXR are activated by several oxysterols or intermediates in the cholesterol homeostasis by coordinately regulating several genes involved in the efflux, transport, and excretion of cholesterol (Tamehiro, et al . , 2005). These receptors have different tissue distributions: while LXRβ is ubiquitously expressed, the expression of LXRα is limited to the liver, kidney, intestine, adipose tissue and macrophages (Lu et al . , 2001). Mice with inactivated LXRβ suffer from adult-onset motor neuron degeneration, associated with lipid accumulation and loss of motor neurons in the spinal cord, together with axonal atrophy and astrogliosis (Andersson et al., 2005). Effects on the survival of midbrain dopaminergic neurons have been observed in null mutant mice, but these effects were attributed to a neurodegenerative process secondary to lipid accumulation in adult brains (Wang et al., 2002).

The present inventors have discovered that LXR ligands and receptors promote neurogenesis. Thus, by regulating the level and/or activity of LXR receptors in cultures or in the brain, the invention allows the promotion of neurogenesis relative to gliogenesis or vice versa 5 and provides for increased neuronal or glial development, differentiation and/or maturation, for example from a stem cell, neural stem cell or dopaminergic precursor or progenitor cell in vitro or in vivo. The invention further allows the increased induction of a neuronal or a glial fate in stem, progenitor,

.0 precursor or neuronal cells in vitro or in vivo and/or increased yield of neurons, in particular dopaminergic neurons, for example relative to glial cells or glial cells relative to neurons. This may be useful, for example, in cell replacement therapies for treating conditions characterised by neuronal loss, damage or dysfunction,

L5 such as Parkinson's disease, stroke, Huntington' s disease and motor neuron disease, in the treatment of proliferative disorders, for example by limiting proliferation of brain tumours and inducing tumour cell differentiation, and for studying signalling events in neurons and the effects of drugs on neurons in vitro, for instance

20 in high throughput screening.

An aspect of the invention provides 'a method of inducing or increasing glial or neuronal development, maturation or differentiation and/or the acquisition of a differentiated phenotype 25 in a neural stem, embryonic stem, progenitor or precursor cell, the method comprising: modulating the amount of liver X receptor signalling in said cell, thereby increasing development, maturation or differentiation 30 in said cell.

Liver X receptor (LXR) signalling may be modulated for example, by increasing or reducing the level and/or activity of an LXR receptor in the cell.

Methods of the invention may be useful in promoting neurogenesis. For example, a method of inducing or increasing neuronal development, maturation or differentiation and/or the acquisition of a neuronal phenotype in a neural stem, embryonic stem, progenitor or precursor cell, may comprise: increasing the amount of liver X receptor signalling in said cell, thereby increasing neuronal development in said cell.

The cell may develop, mature or differentiate into a neuron or an immediate precursor thereof, for example a dopaminergic neuron.

A LXR receptor is a polypeptide is able to form a LXR receptor/ligand complex with a suitable binding partner. Examples of suitable binding partners include LXR ligands. A LXR receptor may be an LXRα or a LXRβ receptor from any mammalian species, for example a mouse LXRα or LXRβ receptor or human LXRα or LXRβ receptor, or a fragment or variant of any one of these.

A fragment or variant of a wild-type LXR receptor sequence as described herein may differ from the wild-type sequence by the addition, deletion, substitution and/or insertion of one or more amino acids, provided the function of modulating development of a neuronal fate, in particular a dopaminegic fate in a stem cell, neural stem cell, embryonic stem cell or neural progenitor or precursor cell is retained. (Monaghan et al. (1999), Bafico et al., (2001); Li et al. (2002); MacDonald et al. (2004); Mao et al . (200Ia) _/ Semenov et al., (2001).

For example, a polypeptide which is a variant of a wild-type sequence may comprise an amino acid sequence which shares greater than about 30% sequence identity with the wild-type sequence, for example human LXRα or LXRβ or one or more domains thereof, greater

than about 40%, greater than about 45%, greater than about 55%, greater than about 65%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%. The sequence may share greater than about 30% similarity with the wild-type sequence, for example human LXRα or LXRβ or one or more domains thereof, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity.

The amino acid sequence of human LXRα (also known as NR1H3; NCBI GenelD: 10062) is available under GenBank reference Swiss protein accession number NP_005684.1 GI: 5031893 and the encoding nucleic acid under reference NM_005693.1 GI: 5031892.

The amino acid sequence of human LXRβ (also known as NR1H2; NCBI GenelD: 7376) is available under GenBank reference NP_009052.3 GI: 85362735 and the encoding nucleic acid under reference NM_007121.3 GI: 85362734.

The amount of liver X receptor signalling in a cell may be increased by treating the cell with an LXR activator. An LXR activator is a compound which increases the amount of LXR signalling in a cell, for example by increasing the level or activity of an LXR receptor. The cell may be treated with the LXR activator in vivo, ex vivo, or in culture.

Suitable LXR activators include LXR ligands . LXR ligands include oxysterols and components in their synthetic pathway, such as (22(R)-OH-cholesterol, 20 (S) -OH-cholesterol, 25-OH-cholesterol, 27- OH-cholesterol; 22-dehydroxycholesterol, 5, 6-24 (s) , 25- diepoxycholesterol 24 (s) , 25-epoxycholesterol; Desmosterol, Zymosterol, Lanosterol, Lathosterol and 7-dehydrocholesterol (See also table 1 in Janowski et al . 1999 PNAS USA 96, 266-277).

Other LXR ligands may include ketosterols and ketocholestenoic acids, such as 3-keto-lithocholic acid, Lathosterone, Lophenone, 4- cholesten-3-one, (25R) 26-OH-4-cholesten-3-one, (25S) 26-OH-4- cholesten-3-one, (25R) 26-3-keto-4-cholestenoic acid and (25S) 26-3- keto-4-cholestenoic acid (Motola et al., 2006, Cell 124, 1209-1223)

Other LXR ligands may include T0901317, T0314407, GW3965 and Acetyl- podocarpic-dimer (APD) .

LXR ligands are described in Millat et al., 2003 Biochimica et Biophysica Acta 1631, 107-118; Yang et al . , 2006 (JBC on line, manuscript M603781200/ Janowski et al . , 1999 PNAS USA 96, 266-277,

Janowski et al . 1996 Nature 383, 728-731 and Motola et al . 2006 Cell 124, 1209-1223.

Suitable LXR activators also include retinoid X receptor (RXR) ligands such as 9-cis retinoic acid, SR11237, (Gendimenico, G. J., et al., (1994) J Invest Dermatol 102(5), 676-80), 9-cis retinol or docosahexanoic acid (DHA), LG849 (Mata de Urquiza et al . , 2000), or LG100268 RXR ligands may bind to RXR in LXR/RXR heterodimers .

An LXR activator may increase the amount of LXR signalling in a cell by reducing the level or activity of a negative LXR factor, which reduces the amount of LXT signalling in a cell. LXR activators may, for example, include nucleic acids, such as anti-sense or siRNA molecules, which reduce the expression of a negative LXR factor. Negative LXR factors whose level or activity may be reduced by an LXR activator may include repressors, such as NcoR and Smart, which reduce the expression of LXR receptors or ligands, antagonists, such as 22-S-OH-cholesterol, which inhibit the activity of LXR receptors or ligands, and catabolic enzymes which increase the turnover of LXR receptors or ligands and reduce the amount of LXR receptors or

ligands in the cell, such as enzymes involved in ubiquitination and sumoylation pathways.

Suitable LXR activators may increase the amount or activity of a positive LXR factor which increases the amount of LXR signalling in a cell. Positive LXR factors may include activators which increase the expression of LXR receptors or ligands, co-factors which increase the activity of LXR receptors or ligands, and biosynthesis enzymes which increase the rate of biosynthesis of LXR receptors or ' ligands and thereby increase the amount of LXR receptors or ligands in the cell, for example, cytochrome P-450 enzymes (cyp39 or cyp46) for oxysterols (Rusell 2000, BBA 1529, 126-135) and cyp27 for ketocholestenoic acids (Motola et al . , 2006).

Suitable LXR activators also include LXR receptors and nucleic acids encoding LXR receptors. Suitable LXR receptors and encoding nucleic acid are described in more detail above.

In some preferred embodiments, a cell may be treated with two or more LXR activators, for example a LXR ligand and a LXR receptor.

Other aspects of the invention provide the use of an LXR activator as described above in a culture medium for supporting neurogenesis of neural stem, embryonic stem, progenitor or precursor cell and a culture medium for supporting neurogenesis of neural stem, embryonic stem, progenitor or precursor cell comprising an LXR activator as described above.

Other components of culture media for use in the differentiation of precursor cells are well known in the art.

Methods of the invention may also be useful in promoting gliogenesis. A method of inducing or increasing glial development, maturation or differentiation and/or the acquisition of a glial

phenotype in a neural stem, embryonic stem, progenitor or precursor cell, may comprise: reducing the amount of liver X receptor signalling in said cell, thereby increasing glial development in said cell.

The cell may develop, mature or differentiate into a glial cell, for example an astrocyte, oligodendrocyte, microglia, satellite cell or Schwann cell.

The amount of liver X receptor signalling in a cell may be reduced by treating the cell with an LXR suppressor. An LXR suppressor is a compound which decreases the amount of LXR signalling in a cell, for example by reducing the level or activity of an LXR receptor. The cell may be treated with the LXR suppressor in vivo> ex vivo, or in culture.

Suitable LXR suppressors include anti-sense or RNAi nucleic acid molecules which target the LXRalpha or LXRbeta. LXR suppressor molecules are described in more detail below.

In some preferred embodiments, a LXR suppressor may selectively down-regulate the expression of LXRα or LXRβ . Down regulation may occur, for example, through RNA interference (RNAi) .

Small RNA molecules may be employed to regulate gene expression. These include targeted degradation of mRNAs by small interfering RNAs (siRNAs) , post transcriptional gene silencing (PTGs) , developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted transcriptional gene silencing.

A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific

chromosomal loci has also been demonstrated. Double-stranded RNA (dsRNA) -dependent post transcriptional silencing, also known as RNA interference (RNAi) , is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 19-nt or 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.

In the art, these RNA sequences are termed "short or small interfering RNAs" (siRNAs) or "microRNAs" (miRNAs) depending in their origin. Both types of sequence may be used to down-regulate gene expression by binding to complimentary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNA are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro-interfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.

Accordingly, the present invention provides the use of these sequences as LXR suppressors for downregulating the expression of

LXRα or LXRβ and thereby reducing the amount of LXR signalling in a cell.

The siRNA ligands are typically double stranded and, in order to optimise the effectiveness of RNA mediated down-regulation of the function of a target gene, it is preferred that the length of the siRNA molecule is- chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of

the mRNA target and so that the siRNA is short enough to reduce a host response.

miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA -sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a partially double stranded RNA segment. The design of microRNA sequences is discussed on John et al, PLoS Biology, 11(2), 1862- 1879, 2004.

Typically, the RNA ligands intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof) , more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the invention employing double-stranded siRNA, the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo) nucleotides, typically a UU of dTdT 3' overhang. Based on the known LXRα and LXRβ sequences and the disclosure provided herein, the skilled person can readily design of suitable siRNA and miRNA sequences, for example using resources such as Ambion's siRNA finder, see http: //www. ambion.com/techlib/misc/siRNA_finder. html. siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors) . In a preferred embodiment the siRNA is synthesized synthetically .

Longer double stranded RNAs may be processed in the cell to produce siRNAs (see for example Myers (2003) Nature Biotechnology 21:324-

328) . The longer dsRNA molecule may have symmetric 3' or 5 ' overhangs, e.g. of one or two (ribo) nucleotides, or may have blunt ends. The longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most preferably, the longer dsRNA molecules are 27 nucleotides in length. dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al . , Genes and Dev., 17, 1340-5, 2003).

Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. A shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression. In a preferred embodiment the shRNA is produced endogenously (within a cell) by transcription from a vector. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human Hl or 7SK promoter or a RNA polymerase II promoter. Alternatively, the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell.

Preferably, the shRNA molecule comprises a partial sequence of LXRα and LXRβ mRNA. Preferably, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilise the hairpin structure.

siRNA molecules, longer dsRNA molecules or miRNA molecules may be made recombinantly by transcription of a nucleic acid sequence,

preferably contained within a vector. Preferably, the siRNA molecule, longer dsRNA molecule or miRNA molecule comprises a partial sequence of the LXRα or LXRβ mRNA.

5 In one embodiment, the siRNA, longer dsRNA or miRNA is produced endogenously (within a cell) by transcription from a vector. The vector may be introduced into the cell in any of the ways known in the art. Optionally, expression of the RNA sequence can be regulated using a tissue specific promoter. In a further

.0 embodiment, the siRNA, longer dsRNA or miRNA is produced exogenously (in vitro) by transcription from a vector.

In one embodiment, the vector may comprise a nucleic acid sequence in both the sense and antisense orientation, such that, when

L5 expressed as RNA, the sense and antisense sections will associate to form a double stranded RNA. Preferably, the vector comprises LXRα or LXRβ nucleic acid sequences; or variants or fragments thereof. In another embodiment, the sense and antisense sequences are provided on different vectors.

20

Alternatively, siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques which are known in the art. Linkages between nucleotides may be phosphodiester bonds or alternatives, for example, linking groups of the formula P(O)S, 5 (thioate); P(S)S, (dithioate) ; P (O) NR ¹ 2; P(O)R'; P(O)OR6; CO; or

CONR '2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S- .

Methods relating to the use of RNAi to silence genes in C. elegans, 30 Drosophila, plants, and mammals are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference < 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al . , Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et

al., Science 286, 950-952 (1999); Hammond, S. M., et al . , Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al . , Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619, and Elbashir S M, et al . , 2001 Nature 411:494-498).

Suitable RNAi molecules for down-regulation of LXRα or LXRβ may comprise a sequence having 85% or more, 90% or more, 95% or more or 100% sequence identity with a contiguous sequence of 10 to 40 nucleotides from the LXRα or LXRβ mRNA sequence.

A cell may be treated with a compound, such as an LXR activator or suppressor, by means of provision of purified and/or isolated compound to a culture comprising the stem, progenitor or precursor cell, or to such a cell in vivo.

In embodiments in which the compound is a peptide or polypeptide, a cell may be treated with the compound by introducing one or more copies of the compound or the encoding nucleic acid into the cell. Methods of transforming cells with nucleic acid and introducing proteins into cells are described further below.

Contacting with a compound, such as an LXR activator or suppressor may be by means of providing in vivo or within a culture comprising the stem, progenitor or precursor cell or neuronal cell, a cell that produces the compound. The cell that produces the compound may be a recombinant host cell that produces the compound either directly by recombinant expression or indirectly, by recombinant expression of the appropriate synthetic enzymes. For example, cytochrome P-450 enzymes (cyp39 or cyp46) may be expressed to produce oxysterol LXR ligands (Rusell 2000, BBA 1529, 126-135) and cyp27 may be expressed to produce ketocholestenoic acid LXR ligands (Motola et al . 2006).

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

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

The methods provided herein may be applied to the induction of neuronal or glial fates in neural stem cells or neural progenitor or precursor cells and also other stem, progenitor or precursor cells which are not committed to a neural fate and may, for example, be capable of giving rise to two or more daughter stem cells associated with different developmental systems. Examples of such cells include embryonic stem cells, in particular non-human embryonic stem cells, and stem cells associated with non-neural systems. The methods may be applied to stromal or hematopoietic stem cells and/or proliferative cells from the epidermis. Hematopoietic cells may be

collected from blood or bone marrow biopsy. Stromal cells may be collected from bone marrow biopsy. Epithelial cells may be collected by skin biopsy or by scraping e.g. the oral mucosa. Since a neuronal phenotype is not a physiological in vivo fate of these stem, progenitor or precursor cells, the inductive process may be referred to as trans-differentiation, or de-differentiation and neural re-differentiation.

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

In preferred embodiments of the present invention, adult stem/progenitor/precursor cells are used, in vitro, ex vivo or in vivo. This requires a consenting adult (e.g. from which the cells are obtained) and approval by the appropriate ethical committee. If a human embryo/fetus is used as a source, the human embryo is one that would otherwise be destroyed without use, or stored indefinitely, especially a human embryo created for the purpose of IVF treatment for a couple having difficulty conceiving. IVF generally involves creation of human embryos in a number greater than the number used for implantation and ultimately pregnancy. Such spare embryos may commonly be destroyed. With appropriate consent from the people concerned, in particular the relevant egg donor and/or sperm donor, an embryo that would otherwise be destroyed can be used in an ethically positive way to the benefit of

sufferers of severe neurodegenerative disorders such as Parkinson' s disease. The present invention itself does not concern the use of a human embryo in any stage of its development. As noted, the present invention minimizes the possible need to employ a material derived directly from a human embryo, whilst allowing for development of valuable therapies for terrible diseases. Any therapeutic interventions based on the present invention must also be performed according to the relevant national laws and ethical guidelines.

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

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

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

Preferably, the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is mitotic and/or capable of self-renewal when it is treated with the LXR activator or suppressor as described herein.

Neuronal development may be characterised by the onset of a neuronal phenotype which includes expression of a nuclear receptor of the Wurrl subfamily, such as Nurrl and other markers such as Lmxla, Lmxlb, engrailed-1 or -2, b-tubulin III, pitx3, DAT or c-ret. A cell treated as described herein may express such markers.

Any method of treating a stem or neural cell according to the invention can be used in combination with any another such method, either together or sequentially. Examples of other such methods are described in more detail below.

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

In a method of inducing a glial fate wherein the cells are treated with a LXR suppressor, a majority of the cells may be induced to adopt a glial fate. In preferred embodiments, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% of the stem and/or progenitor cells may be induced to a glial fate.

Compounds which are peptides, polypeptides or proteins may be introduced via protein transduction or expressed from encoding nucleic acid either in situ in a stem, or neural stem, precursor or progenitor cell or neuronal cell or in' vitro in an expression system prior to isolation and purification. In some embodiments, protein transduction may be preferred, since the absence of genetic modification may facilitate clinical application.

Transformed nucleic acid, for example encoding an LXR activator or suppressor, may be contained on an extra-genomic vector or it may be incorporated, preferably stably, into the genome. It may be operably-linked to a promoter which drives its expression above basal levels in stem cells, or neural stem, precursor or progenitor cells, or neuronal cells, as is discussed _. in more detail below.

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

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

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

For further details see, for example, Molecular Cloning-: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds . , John Wiley & Sons, 1992 or later edition.

Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well-known in the art. Clones may also be identified or further investigated by binding studies, e.g. by Southern blot hybridisation.

Nucleic acid encoding a LXR activator or suppressor may be integrated into the genome of the host stem, neural stem, progenitor, precursor or neural cell. Integration may be promoted by

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

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

Suitable vectors and techniques for in vivo transformation of stem cells, or neural stem, precursor or progenitor cells or neuronal cells with nucleic acid encoding a LXR activator or suppressor are

well known to those skilled in the art. Suitable vectors include adenovirus, adeno-associated virus papovavirus, vaccinia virus, herpes virus, lentiviruses and retroviruses. Disabled virus vectors may be produced in helper cell lines in which genes required for 5 production of infectious viral particles are expressed. Suitable helper cell lines are well known to those skilled in the art. By way of example, see: Fallaux, F.J., et al . , (1996) Hum Gene Ther 7(2), 215-222; Willenbrink, W., et al., (1994) J Virol 68(12), 8413-8417; Cosset, F. L., et al . , (1993) Virology 193(1), 385-395; Highkin,

.0 M.K., et al., (1991) Poult Sci 70(4), 970-981; Dougherty, J. P., et al., (1989) J Virol 63(7), 3209-3212; Salmons, B., et al . , (1989) Biochem Biophys Res Commun 159(3), 1191-1198; Sorge, J., et al., (1984) MoI Cell Biol 4(9), 1730-1737; Wang, S., et al . , (1997) Gene Ther 4(11), 1132-1141; Moore, K.W., et al . , (1990) Science

L5 248(4960), 1230-1234; Reiss, CS. , et al . , (1987) J Immunol 139(3), 711-714. Helper cell lines are generally missing a sequence which is recognised by the mechanism which packages the viral genome. They produce virions which contain no nucleic acid. A viral vector which contains an intact packaging signal along with the gene or

20 other sequence to be delivered (e.g. LXR activator coding sequence or LXR suppressor) is packaged in the helper cells into infectious virion particles, which may then be used for gene delivery to stem cells, or neural stem, precursor or progenitor cells or neuronal cells . 5

As an alternative or addition to increasing transcription and/or translation of an endogenous LXR activator, such as an LXR receptor, expression of LXR activator above basal levels may be caused by introduction of one or more extra copies of the LXR activator into

30 the stem, neural stem, precursor, progenitor or neural cell by microinjection or other carrier-based or protein delivery system including cell penetrating peptides, i.e. TAT, transportan, Antennapedia penetratin peptides (Lindsay 2002) .

The present invention allows for generation of large numbers of neurons, in particular dopaminergic neurons. These neurons may be used as source material to replace cells which degenerate or are damaged or lost in Parkinson's disease.

The present invention also allows for generation of large numbers of glial cells. These glial cells may be useful as source material for in vitro or transplantation purposes. For example, glial cells produced as described herein may be co-cultured with stem cells to induce differentiation into DA neurons, or may be co-grafted with neurons or stem cells in vivo.

In methods of the invention, the cell may additionally be contacted with one or more agents selected from: basic fibroblast growth factor (bFGF) ; epidermal growth factor (EGF) ; and an activator of the retinoid X receptor (RXR), e.g. the synthetic retinoid analog SR11237, (Gendimenico, G. J., et al . , (1994) J Invest Dermatol 102(5), 676-80), 9-cis retinol or docosahexanoic acid (DHA) or LG849 (Mata de Urquiza et al., 2000) ; a member of the Wnt family of ligands, including Wnt-1, -3a and -5a (Catello-Branco et al., 2003); an upstream regulator of Wnts, such as Msx-1 (Her and Abate-Shen, Biochemical And Biophysical Research Communications 227, 257-265 (1996); Shang et al . , Proc. Natl. Acad. Sci. USA 91, 118-122 (1994); or GSK3 inhibitors or b-catenin (Catello-Branco et al., 2003); or Ngn2 (Kelle et al., 2006), or Nurrl (Wagner et al., 1999) or other factors such as Pitx3, Lmxla, Lmxlb, Msxl, Foxa2 or Shh. Treating cells in accordance with the invention with one or more of these agents may be used to increase the proportion of the stem, progenitor or precursor cells, which adopt a neuronal, preferably a dopaminergic fate, or enhance dopaminergic induction or differentiation in a neuronal cell. The method of inducing a neuronal, preferably a dopaminergic fate or enhancing neuronal, preferably dopaminergic, induction or differentiation in a neuronal cell in accordance with the present invention may include contacting

the cell with a member of the FGF family of growth factors, e.g. FGF4, FGF8 or FGF20, for example in a pre-treatment step.

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

In some preferred embodiments, the cell may additionally be contacted with a member of the Wnt family of ligands, including Wnt polypeptides such as Wnt-1, -2, -3a, 7a and -5a, or an activator or upstream regulator of Wnts, such as Msx-1.

A cell may be contacted with a Wnt ligand or activator by adding purified and/or recombinant Wnt ligand or activator to a culture comprising the neural cell, or to such a cell in vivo. Contacting a cell with a Wnt ligand or activator may comprise introducing one or more copies of Wnt nucleic acid into the cell, and allowing the protein to be expressed, or introducing the protein itself into the cell. Methods of transforming cells with nucleic acid and introducing proteins into cells are described herein. Contacting with a Wnt ligand or activator may be by means of providing in vivo or within a culture comprising the neural cell a cell that produces the Wnt ligand or activator. The cell that produces the Wnt ligand or activator may be a recombinant host cell that produces Wnt ligand or activator by recombinant expression. A co-cultured host cell may be transformed with nucleic acid encoding a Wnt ligand or activator, and/or the co-cultured cell may contain introduced Wnt ligand or activator. The nucleic acid or protein may be introduced into the cell in accordance with available techniques in the art, examples of which are described herein. The co-cultured or host cell may be another neural cell e.g. a stem, neural stem, progenitor, precursor or neuronal cell. Contact with a Wnt ligand or activator may also be by means of up regulating its expression in the cell or by down regulating or inhibiting an inhibitor molecule of the Wnt ligand or activator. Thus contact with a Wnt ligand or activator may arise by

decreasing expression or activity of Wnt-interacting molecules, such as SFRP, WIF, dkk or Cerberus (Martinez Arias et al . , 1999; and the Wnt home page at http://www.stanford.edu/~rnusse/wntwindow.html or findable using any web browser) .

As used herein, a "Wnt polypeptide", "Wnt glycoprotein" or "Wnt ligand" refers to a member of the Wingless-irit family of secreted proteins that regulate cell-to-cell interactions. Wnts are highly conserved from Drosophila and Caenorhabditis elegans, to Xenopus, zebra fish and mammals. The 19 Wnt proteins currently known in mammals bind to two cell surface receptor types: the seven transmembrane domain Frizzled receptor family, currently formed by 10 receptors, and the L_ow density lipoprotein-receptor irelated p_roteins (LRP) 5 and 6 and the kremen 1 and 2 receptors. The signal conveyed by Wnts is transduced via three known signalling pathways: (1) the so called canonical signalling pathway, in which GSK3 beta is inhibited, does not phosphorylate beta-catenin, which is then not degraded and is translocated to the nucleus to form a complex with TCF and activate transcription of Wnt target genes. (2) the planar polarity and convergence-extension pathway, via Jnk. (3) and the inositol 1,4,5 triphosphate (IP3) /calcium pathway, in which calcineurin dephosphorylates and activates the nuclear factor of activated T cells (NF-AT) (Saneyoshi et al., 2002). For review see the Wnt home page, findable on the web using any available browser (currently at www.stanford.edu/~rnusse/wntwindow.html). Other co- receptors involved in Wnt signaling include the tyrosine kinase receptor Rorl and Ror2 (Oishi I et al . , 2003), the derailed/RYK receptor family (Yoshikawa et al., 2003), which encode catalytically inactive receptor tyrosine kinases.

The Wnt ligand may be a Wntl polypeptide or a variant thereof. Human Wntl amino acid sequence is available under GenBank reference Swiss protein accession number P04628 and encoding nucleic acid under reference X03072.1 for DNA and NM 005430.2 for RNA.

The Wnt ligand may be a Wnt5a polypeptide or a variant thereof. Human Wnt5a amino acid sequence is available under GenBank reference Swiss protein accession number P41221 and encoding nucleic acid under references AI634753.1 AK021503 L20861 L20861.1 and U39837.1 for DNA and NM_003392 for RNA.

Although not preferred for generation of DA neurons, the Wnt ligand may be a Wnt3a polypeptide or a variant thereof. A Wnt3a polypeptide may be used to maintain the proliferation or self- renewal of stem/progenitor cells and/or allow or induce their differentiation into other, i.e. non-dopaminergic, neuronal phenotypes. Wnt3a decreases the number of Nurr-1 expressing progenitors that give rise to DA neurons. However, since the total number of neurons is not decreased other neuronal phenotypes may be produced, e.g. dorsal midbrain phenotypes, including serotonergic neurons. Loss of serotonergic neurons is associated with depression, so neurons generated by methods comprising use of a Wnt3a ligand, and/or a Wnt3a ligand itself, may be used in therapies e.g. of depression.

Human Wnt3a amino acid sequence is available under GenBank reference SwissProt Accession No.P56704 and encoding nucleic acid under reference AB060284 AB060284.1 AK056278 AK056278.1 for DNA and NMJ333131 for mRNA.

A wild-type Wnt ligand may be employed, or a variant or derivative, e.g. by addition, deletion, substitution and/or insertion of one or more amino acids, provided the function of enhancing development of a dopaminergic neuronal fate in a stem cell, neural stem cell or neural progenitor or precursor cell is retained.

A method as described herein in which a neuronal fate is induced in a stem, neural stem or progenitor or precursor cell or there is

increased neuronal, preferably dopaminergic, induction, maturation or differentiation in a neuronal cell, may include detecting a marker for the neuronal fate, β-tubulin III (TuJl) is one marker of the neuronal fate (Menezes, J. R., et al . , (1994) J Neurosci 14(9), 5 5399-5416). Other neuronal markers include neurofilament and MAP2. If a particular neuronal phenotype is induced, the marker should be specific for that phenotype. For the dopaminergic fate, expression of tyrosine hydroxylase (TH) , aromatic L-amino acid decarboxylase (AADC) , dopamine transporter (DAT) and dopamine receptors may be

LO detected e.g. by immunoreactivity or in situ hybridization.

Tyrosine hydroxylase is a major marker for DA cells. Contents and/or release of dopamine and metabolites may be detected e.g. by High Pressure Liquid Chromatography (HPLC) (Cooper, J. R., et al., The Biochemical Basis of Neuropharmacology, 7th Edition, (1996)

L5 Oxford University Press) . The absence of Dopamine β hydroxylase and GABA or GAD (in the presence of TH/dopamine/DAT) is also indicative of dopaminergic fate. Additional markers include Nurrl, Aldehyde dehydrogenase type 2 (ADH-2), GIRK2, Lmxla, Lmxlb, engrailed -1 and -2, and Pitx3.

>0

A method as described herein in which a glial fate is induced in a stem, neural stem or progenitor or precursor cell or there is increased glial induction, maturation or differentiation in a cell, may include detecting a marker for the glial fate. Glial markers

15 include glial fibrilary acidic protein (GFAP) , vimentin, RC2, NG2, A2B5, 04, RIP, Brain lipid binding protein (BLBP) and myelin basic protein (MBP) .

Detection of a marker may be carried out according to any method 50 known to those skilled in the art. The detection method may employ a specific binding member capable of binding to a nucleic acid sequence encoding the marker, the specific binding member comprising a nucleic acid probe hybridisable with the sequence, or an immunoglobulin/antibody domain with specificity for the nucleic acid

sequence or the polypeptide encoded by it, the specific binding member being labelled so that binding of the specific binding member to the sequence or polypeptide is detectable. A "specific binding member" has a particular specificity for the marker and in normal conditions binds to the marker in preference to other species. Alternatively, where the marker is a specific mRNA, it may be detected by binding to specific oligonucleotide primers and amplification in e.g. the polymerase chain reaction.

Nucleic acid probes and primers may hybridize with the marker under stringent conditions. Suitable conditions include, e.g. for detection of marker sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55C in 0.1X SSC, 0.1% SDS. For detection of marker sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65°C in 0.25M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60 ⁰ C in 0.1X SSC, 0.1% SDS.

Another aspect of the invention provides a cell produced in accordance with any one of the methods disclosed herein. In some embodiments, the cell may be a neuron or an immediate precursor thereof. The cell may have a primitive neuronal phenotype and may be capable of giving rise to a plurality of distinct neuronal phenotypes. Alternatively, the neuron may have a particular- neuronal phenotype. In preferred embodiments, the neuron has a dopaminergic phenotype. In other embodiments, the cell may be a glial cell or an immediate precursor thereof. The cell may have a primitive glial phenotype and may be capable of giving rise to a plurality of distinct glial phenotypes. Alternatively, the glial cell may have a particular glial phenotype, for example an astrocyte, oligodendrocyte or Schwann cell phenotype.

A cell produced as described herein, such as a neuron or immediate precursor, may be treated with a factor with neuroprotective or neuroregenerative properties, or transduced with a factor with such properties or contain nucleic acid encoding a factor with neuroprotective or neuroregenerative properties operably linked to a promoter which is capable of driving expression of the factor in the cell. The promoter may be an inducible promoter, e.g. the TetON chimeric promoter, so that any damaging over-expression may be prevented. The promoter may be associated with a specific neuronal phenotype, e.g. the TH promoter, the Nurrl promoter, the dopamine transporter promoter or the Pitx3 promoter.

The factor may be such that its expression renders the cell independent of its environment, i.e. such that its survival is not dependent on the presence of one or more factors or conditions in e.g. the neural environment into which it is to be implanted. By way of example, the cell may contain a factor or protein or nucleic acid encoding one or more of the neuroprotective or neuroregenerative molecules described below operably linked to a promoter that is capable of driving expression of the molecule in the cell.

In addition or alternatively, the factor or expression of the encoded molecule may function in neuroprotection or neuroregeneration of the cellular environment surrounding that neuron. In this way, the neuron may be used to deliver molecules with neuroprotective and neuroregenerative properties in a cell therapy approach (when treated with factors, proteins or protein transduction) or as a less preferred option as a combined cell and gene therapy approach.

Examples of molecules with neuroprotective and neuroregenerative properties include:

(i) neurotropic factors able to compensate for and prevent neurodegeneration. One example is glial-derived neurotropic growth factor (GDNF) which is a potent neural survival factor, promotes sprouting from dopaminergic neurons and increases tyrosine

5 hydroxylase expression (Tomac, et al. (1995) Nature, 373, 335-339; Arenas, et al . , (1995) Neuron, 15,1465-1473). By enhancing axonal elongation GDNF, GDNF may increase the ability of the neurons to inervate their local environment. Other neurotropic molecules of the GDNF family include Neurturin, Persephin and Artemin .

0 Neurotropic molecules of the neurotropin family include nerve growth factor (NGF) , brain derived neurotropic factor (BDNF) , and neurotropin-3, -4/5 and -6. Other factors with neurotrophic activity include members of the FGF family for instance FGF2, 4, 8 and 20; members of the Wnt family, including Wnt-1, -2, -5a, -3a and 7a;

5 members of the BMP family, including BMP2, 4, 5 and 7, nodal, activins and GDF; and members of the TGFalpha/beta family.

(ii) anti-apoptotic molecules. Bcl2 which plays a central role in cell death. Over-expression of Bcl2 protects neurons from naturally 0 occurring cell death and ischemia (Martinou, et al . , (1994) Neuron, 1017-1030) . Another anti-apoptotic molecule specific for neurons is BcIX-L.

(iii) axon regenerating and/or elongating and/or guiding molecules :5 which assist the neuron in innervating and forming connections with its environment, e.g. ephrins or semaphorins, netrins, wnts. Ephrins define a class of membrane-bound ligands capable of activating tyrosine kinase receptors. Ephrins have been implicated in neural development (Irving, et al., (1996) Dev. Biol., 173, 26- :0 38; Krull, et al . , (1997) Curr. Biol. 7, 571-580; Frisen, et al., (1998) Neuron, 20, 235-243; Gao, et al . , (1996) PNAS, 93, 11161- 11166; Torres, et al . , (1998) Neuron, 21, 1453-1463; Winslow, et al., (1995) Neuron, 14, 973-981; Yue, et al . , (1999) J Neurosci 19(6), 2090-2101.

(iv) transcription factors, e.g. the homeobox domain protein Ptx3 (Smidt, M. P., et al . , (1997) Proc Natl Acad Sci USA, 94(24), 13305- 13310), Lmxlb, Pax2, Pax5, Pax8, or engrailed 1 or 2 (Wurst and Bally-Cuif, 2001; Rhinn and Brand, 2001) ; or upstream regulators of Wnts, including Msx-1 (Her and Abate-Shen, 1996; Shang et al . , 1994); or beta-catenin (Catello-Branco et al., 2003); or Nurrl (Wagner et al., 1999); or neurogenic genes of the basic helix-loop- helix family, such as Ngn2 (Kelle et al . , 2006, Anderson et al., 2006, Development) or Msxl or Lmxla (Anderson et al . , Cell).

A neuron may be substantially free from one or more other cell types, e.g. from stem, neural stem, precursor or progenitor cells or from glial cells.

A glial cell may be substantially free from one or more other cell types, e.g. from stem, neural stem, precursor or progenitor cells or from neurons .

Neurons and glial cells may be separated from neural stem or progenitor cells using any technique known to those skilled in the art, including those based on the recognition of extracellular epitopes by antibodies and magnetic beads or fluorescence activated cell sorting (FACS) . By way of example, antibodies against extracellular regions of molecules found on stem, neural stem, precursor or progenitor cells but not on neurons may be employed. Such molecules include Notch 1, CD133, SSEAl, promininl/2, RPTPβ/phosphocan, and the glial cell line derived neurotrophic factor receptors GFR alphas or NCAM. Stem cells bound to antibodies may be lysed by exposure to complement, or separated by, e.g. magnetic sorting (Johansson et al., (1999) Cell, 96, 25-34). If antibodies which are xenogeneic to the intended recipient of the neurons are used, then any e.g. stem, neural stem or progenitor or precursor cells which escape such a cell sorting procedure are

labelled with xenogeneic antibodies and are prime targets for the recipient's immune system. Alternatively, cells that acquire the desired phenotype could also be separated by antibodies against extracellular epitopes or by the expression of transgenes including 5 fluorescent proteins under the control of a cell type specific promoter. By way of example dopaminergic neurons could be isolated with fluorescent proteins expressed under the control of TH, DAT, Ptx3 or other promoters specifically used by midbrain dopaminergic neurons .

.0

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

L5

Negative selection may be used to enrich for target cells, for example, glial cells or neurons, such as dopaminergic neurons. For example, to select for dopaminergic neurons, selective neurotoxins for non-DA neurons may be used, for instance 5-7-dihydroxytryptamine

20 (to eliminate serotoninergic neurons) , or antibodies coupled to saponin or a toxin or after addition of complement, for instance antibodies against GABA transporter (to eliminate GABAergic neurons) . Methods of the invention may comprise additionally treating or contacting a neural stem, progenitor or precursor cell, 5 or other stem or neural cell with a negative selection agent, preferably in vitro, e.g. by adding the negative selection agent to an in vitro culture containing the cell, or by culturing the cell in the presence of the negative selection agent. A negative selection agent selects against cell types other than the desired cell

30 type(s). For example, where the invention relates to promoting, enhancing or inducing a dopaminergic neuronal phenotype, the negative selection agent may select against cells other than DA neurons and cells that develop into DA neurons such as stem cells and neural stem, precursor and progenitor cells. Thus, the negative

selection agent may select against differentiated cells with a non- DA phenotype, such as non-DA neurons. The negative selection agent may reduce or prevent proliferation of and/or kill cells other than the desired cell type(s). The negative selection agent may be a selective neurotoxin that reduces the population of neurons other than DA neurons. For example, the negative selection agent may be 5-7-dihydroxytryptamine (to reduce serotoninergic neurons) . The negative selection agent may be an antibody or antibody fragment specific for a non-DA neuron, wherein the antibody or antibody fragment (e.g. scFv or Fab) is a blocking antibody or is coupled to saponin or to a toxin. For instance, the antibody may be a blocking antibody against FGF-4 (to reduce serotonin neurons) , or an antibody specific for GABA transporter coupled to a toxin (to reduce GABAergic neurons) .

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

The methods described herein may be useful in producing neurons or the immediate precursors thereof for therapeutic applications. The ability to promote neuronal development, differentiation and/or maturation of stem cells or neural stem, progenitor or precursor cells, or neurons prior to and/or following transplantation, may be useful, for example, in ameliorating the bias of transplanted stem cells to differentiate into neuronal fates when grafted in the adult brain.

The stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell is treated in vitro or ex vivo as described above and then administered an individual, for example an individual having a condition associated with neuronal loss, damage or dysfunction, such as Huntington's disease, Parkinson's disease, a parkinsonian syndrome, neuronal loss or a neurodegenerative disease.

Other conditions suitable for treatment as described herein include degeneration in or damage to the spinal cord and/or cerebral cortex, or other regions of the nervous system, for example stroke or motor neuron disease.

A method of treating a condition associated with neuronal loss, damage or dysfunction in an individual, may comprise (a) treating a cell using a method of increasing neuronal development as described above, and

(b) administering the treated cell, or a descendent of the cell, to the individual.

The treated cell may be administered to an individual, for example by implanting the treated cell into the brain of the individual. Suitable grafting techniques are known in the art. In some embodiments, a neuron or immediate precursor may be administered along with a glial cell produced by methods of increasing glial development described herein. Implanted cells can replace lost neural tissue and thereby ameliorate a condition associated with neuronal loss, damage or dysfunction, or prevent or slow the loss of neural tissue or neurodegeneration in an individual.

The stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell may be in the brain and targeted by the treatment or may be endogenous to the individual to be treated by promoting neurogenesis as described herein, but previously obtained from that individual, or a descended from tissue or cells previously obtained from that individual. An endogenous cell has the same genetic material as cells of the individual being treated, thus limiting problems of rejection by the immune system. Alternatively, the stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell may be exogenous to the individual. Sources of stem cells and neural cells that may be treated or used in a method of the invention are described elsewhere herein. Immune rejection in these cells can be prevented by somatic cell nuclear transfer (SCNT) where a cell nuclei form the host is reprogrammed by injection into the cytoplasm of a stem cell from the donor, or by conventional immunosuppressive therapy.

Other aspects of the invention provide a neuron and/or a glial cell produced in accordance with the present methods for treatment of an individual as described herein, and the use of a neuron and/or a glial cell produced in accordance with the present methods in the manufacture of a medicament for treatment of an individual. The medicament may be for implantation into the brain of the individual, for example for treatment of a condition associated with neuronal loss, damage or dysfunction, such as Huntington' s disease,

Parkinson's disease, a parkinsonian syndrome, neuronal loss or a neurodegenerative disease. The cells can be injected in the substantia nigra or midbrain region or in the target regions of dopaminergic neurons, such as the striatum, which is the region currently preferred.

Materials and methods described herein may be used to treat cells post-transplantation, i.e. in situ in the individual to be treated. Post-implantation treatments may be performed using methods of the

promoting neurogenesis as described herein whether or not the implanted cells were treated by methods of the invention prior to being implanted. The stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell may be treated in an individual in situ with a LXR activator, as described herein.

A method of treating a condition associated with neuronal damage, loss or dysfunction in an individual may comprise; treating a stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell using a method of promoting neuronal development as described herein, wherein the cell is in situ in the brain of the individual.

The stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell may be accompanied by a glial cell produced by the present methods .

A condition associated with neuronal damage, loss or dysfunction individual may include Huntington' s disease, Parkinson's disease, a parkinsonian syndrome, neuronal loss, neurodegenerative disease, and degeneration in or damage to the spinal cord and/or cerebral cortex, or other regions of the nervous system, for example stroke or motor neuron disease, as described above.

A stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell may be treated by administering an LXR activator locally, by infusion of the factor or protein transduction, or by administration of an LXR ligand to the individual either locally or systemically. The LXR ligand may, for example, be administered orally or by infusion using standard techniques .

The stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell may be exogenously supplied by grafting or implanting into the individual. In other words, the method may further comprise administering a stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell to the individual and then treating the cell in situ as described above.

While transplantation of neural tissue is an attractive technique for replacing lost neuronal tissue in individuals who have neurodegenerative disorders or neuronal loss, an alternative treatment is direct infusion of compounds required to promote regeneration, repair or guide the development and/or recruitment of stem or progenitor or precursor cells, or the administration of drugs that regulate those functions. The materials and methods described herein may be performed on non-transplanted cells in situ in the brain of an individual to be treated. The treated stem, embryonic stem, neural stem, progenitor or precursor cells or other stem or neural cells are thus endogenous to the individual and naturally occur in the brain of the individual.

Another aspect of the present invention provides a method of treating neurodegenerative disease or neuronal loss in an individual, the method comprising administering to the individual one or more compounds which increase the amount LXR signalling in a cell of the individual.

Another aspect of the invention provides a method of treating a condition associated with aberrant neural cell proliferation in an individual comprising; administering to the individual one or more compounds which increase the amount LXR signalling in a cell of the individual.

Conditions associated with aberrant neural cell proliferation include brain cancer. The methods described herein may increase differentiation and reduce proliferation of tumour cells in the brain of an individual .

Suitable compounds include LXR activators as described herein, for example, LXR ligands and LXR receptors.

LXR activators as described herein may be administered to an individual in a localised manner to the brain or other desired site or may be delivered in a manner in which it reaches the brain or other neural cells. Preferably, the substance or composition is targeted to the neural cells to be treated.

Targeting therapies may be used to deliver the active substance more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require 'too high a dosage, or if it would not otherwise be able to enter the target cells .

Combination treatments are possible. For example, one or more methods of the invention may be performed to prepare cells for administering into an individual. Following administration, the neural cells may be treated in situ according to one or more methods described herein, to promote further proliferation, maturation and/or differentiation of the administered cells.

For example, in situ treatment with Wnt-5a is useful for promoting neuritogenesis of DA neurones. This may promote integration of the implanted cells into the brain. Wnt-5a can also be used as a treatment on cells before their transplantation into the body, although there may be a risk of damage during administration.

The present invention provides in various aspects and embodiments the use of an LXR activator such as an LXR ligand or LXR receptor as described herein, in therapeutic methods comprising administering the LXR activators to an individual to induce, promote or enhance neurogenesis in the brain, preferably dopaminergic neurogenesis, by- acting on either endogenous or on exogenously supplied stem, progenitor or precursor cells, or neuronal cells, and/or to increase the development, maturation or differentiation of neurons, or onset of a neuronal phenotype, or functional output, of dopaminergic neurons, e.g. in treatment of an individual with a Parkinsonian syndrome or Parkinson's disease, Huntington's disease, stroke or motor neuron disease or in the treatment of a disease associated with aberrant neural cell proliferation. A LXR activator may be administered in any suitable composition, e.g. comprising a pharmaceutically acceptable excipient or carrier, and may be used in the manufacture of a medicament for treatment of a condition associated with neuron loss, damage or dysfunction, for example a neurodegenerative disorder, Parkinsonian syndrome or Parkinson's disease, Huntington's disease, stroke or motor neuron disease or for the treatment of a disease associated with aberrant neural cell proliferation. A LXR activator may be administered to or targeted to the central nervous system and/or brain.

The present invention also extends in various aspects to a pharmaceutical composition, medicament, drug or other composition comprising such an LXR activator, such as a LXR ligand or LXR receptor, and a method of making a pharmaceutical composition comprising admixing such an LXR activator with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients, e.g. a neuroprotective molecule, a neuroregenerative molecule, a retinoid, growth factor, astrocyte/glial cell, optionally an astrocyte or other glial cell produced by a method described herein, an anti-apoptotic factor, or

factor that regulates gene expression in stem, progenitor or precursor cells or neuronal cells or in the host brain. Such optional ingredients may render a neuron independent of its environment, i.e. such that its survival is not dependent on the presence of one or more factors or conditions in its environment.

By way of example, the method of making a pharmaceutical composition may include admixing an LXR activator with one or more factors found in the developing ventral mesencephalon. The LXR activator may be admixed with GDNF and/or neurturin (NTN) and/or brain-derived neurotrophic factor (BDNF) .

Another aspect of the invention provides a method of manufacturing a medicament for treating a condition associated with neuronal loss, damage or dysfunction, comprising: (a) treating a stem, embryonic stem, neural stem, progenitor or precursor cell or other stem or neural cell using a method of promoting neuronal development or a method of promoting glial development, as described herein; and

(b) formulating the treated cell, or a descendent of the cell, into a composition comprising a pharmacologically acceptable excipient .

In some embodiments, a cell treated to develop a neuronal phenotype as described herein may be formulated with a cell treated to develop a glial phenotype as described herein to produce a medicament.

The medicament may be for implantation into the brain of an individual .

The present invention extends in various aspects not only to a cell treated as described herein, or a neuron or glial cell produced in accordance with any one of the methods disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a cell, use of such a cell or composition in a

method of medical treatment, a method comprising administration of such a cell or composition to a patient, e.g. for treatment (which may include preventative treatment) of Parkinson's disease or other (e.g. neurodegenerative) diseases, use of such a cell in the manufacture of a composition for administration, e.g. for treatment of Parkinson's disease or other (e.g. neurodegenerative diseases), and a method of making a pharmaceutical composition comprising admixing such a cell with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients, e.g. a neuroprotective molecule, a neuroregenerative molecule, a retinoid, growth factor, astrocyte/glial cell, anti-apoptotic factor, or factor that regulates gene expression in neuronal cells or in the host brain. Such optional ingredients may render the cell independent of its environment, i.e. such that its survival is not dependent on the presence of one or more factors or conditions in its environment. By way of example, the method of making a pharmaceutical composition may include admixing the cell with one or more factors found in the developing ventral mesencephalon. The cell may be admixed with GDNF and/or neurturin (NTN) and/or BDNF.

Pharmaceutical compositions as described herein and for use in accordance with the present invention, may comprise, in addition to the cell and/or LXR activator, a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabiliser, anti-oxidant or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the activity of the neuron. The precise nature of the carrier or other material will depend on the route of administration. The composition may include one or more of a neuroprotective molecule, a neuroregenerative molecule, a retinoid, growth factor, astrocyte/glial cell, or factor that regulates gene expression in stem, neural stem, precursor or progenitor cells or neuronal cells. Such substances may render the neuron independent of its environment as discussed above.

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

The composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection. A composition may be prepared using artificial cerebrospinal fluid.

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

Cells may be implanted into a patient by any technique known in the art (e.g. 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, C.R. ,(1992) New England Journal of Medicine, 327, 1549-1555).

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

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

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

In order to ameliorate possible disadvantages associated with immunological rejection of transplanted cells, stem or progenitor or precursor cells may be isolated from a patient and induced to the desired neuronal phenotype. Cells may then be transplanted to the patient. Advantageously, isolated stem or progenitor or precursor cells may be used to establish cell lines so that large numbers of immunocompatible neuronal cells may be produced. A further option is to establish a bank of cells covering a range of immunological compatibilities from which an appropriate choice can be made for an individual patient. Stem, neural stem, precursor or progenitor cells or neuronal cells derived from one individual may be altered to ameliorate rejection when they or their progeny are introduced

into a second individual. By way of example, one or more MHC alleles in a donor cell may be replaced with those of a recipient, e.g. by homologous recombination.

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

Other aspects of the invention relates to screening methods for identifying compounds useful in promoting neurogenesis, in particular dopaminergic neurogenesis, or gliogenesis

A method of identifying a compound useful in promoting neurogenesis or gliogenesis may comprise:

(a) contacting a test compound with a cell comprising a nucleic acid which comprises an LXR receptor promoter operably linked to a gene,

(b) determining the amount of expression of the gene, and

(c) comparing the amount of gene expression in the presence of the test compound with the amount of gene expression in the absence of the test compound in comparable conditions, wherein an increase in the amount of gene expression indicates that the test compound is able to promote neuronal development and a decrease in the amount of gene expression indicates that the test compound is able to promote glial development.

The promotion of neurogenesis may be characterised by enhancing neuronal development, differentiation and/or maturation or the onset of a neuronal phenotype in a neural stem, embryonic stem, progenitor or precursor cell.

The promotion of gliogenesis may be characterised by enhancing glial development, differentiation and/or maturation or the onset of a glial phenotype in a neural stem, embryonic stem, progenitor or precursor cell. 5

The method may further comprise identifying the test substance as a modulator, for example an enhancer or repressor, of expression of LXR receptor.

.0 λ change in expression of the gene compared with expression of another gene linked to a different promoter indicates specificity of the substance for modulation of the LXR promoter.

The gene may be the LXR receptor gene itself or it may be a .5 heterologous gene, e.g. a reporter gene. A "reporter gene" is a gene whose encoded product may be assayed following expression, i.e. a gene which "reports" on promoter activity. Suitable reporter genes are discussed elsewhere herein.

!0 By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) . The promoter of an LXR receptor gene may comprise one or more fragments of the sequence under the accession number, sufficient to

!5 promote gene expression. The promoter of a gene may comprise or consist essentially of a sequence of nucleotides 5' to the gene in the human chromosome, or an equivalent sequence in another species, such as a rat or mouse. For example, up to 100kb, 50kb, 20kb, 10kb, 5kb, 2kb or lkb of

10 sequence 5' of the transcription start site may be employed. Conveniently, a knock-in approach may be employed in which a reporter or other gene replaces the endogenous LXR receptor gene in a cell. All regulatory elements of the endogenous LXR receptor gene are thus retained.

The level of promoter activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter. The amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction (PCR) .

PCR comprises steps of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerisation. The nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA. Other specific nucleic acid amplification techniques include strand displacement activation, the QB replicase system, the repair chain reaction, the ligase chain reaction and ligation activated transcription. For convenience, and because it is generally preferred, the term PCR is used herein in contexts where other nucleic acid amplification techniques may be applied by those skilled in the art. Unless the context requires otherwise, reference to PCR should be taken to cover use of any suitable nucleic amplification reaction available in the art.

Use of a reporter gene facilitates determination of promoter activity by reference to protein production. The reporter gene preferably encodes an enzyme which catalyses a reaction that produces a detectable signal, preferably a visually detectable signal, such as a coloured product. Many examples are known, including β-galactosidase and luciferase. β-galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectrophotometer to measure absorbance. Fluorescence, for example that produced as a result of luciferase

activity or green or red fluorescent proteins, may be quantified using a spectrophotometer or fluorescent microscopy. Radioactive assays may be used, for instance using chloramphenicol acetyltransferase, which may also be used in non-radioactive assays. The presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof. The binding molecule may be labelled directly or indirectly using any standard technique.

A promoter construct may be introduced into a cell line using any suitable technique to produce a stable cell line containing the reporter construct integrated into the genome. The cells may be grown and incubated with test compounds for varying times. The cells may, for example, be grown in 96 well (or larger) plates to facilitate the analysis of large numbers of compounds. The cells may then be washed and the reporter gene expression analysed. For some reporters, such as luciferase the cells will be lysed then analysed.

Those skilled in the art are aware of a multitude of possible reporter genes and assay techniques which may be used to determine gene activity. For more examples, see Sambrook and Russell 2001.

A method of identifying a compound useful in promoting neurogenesis or gliogenesis may comprise:

(a) contacting an LXR receptor and a test compound;

(b) determining the LXR receptor activity, and (c) comparing the LXR receptor activity in the presence of the test compound with the LXR receptor activity in the absence of the test compound in comparable reaction medium and conditions, wherein an change in LXR receptor activity in the presence of the test compound is indicative that the test compound is useful in promoting neuronal or glial development.

An increase in LXR receptor activity in the presence of the test compound is indicative that the test compound is useful in promoting neuronal development. 5

A decrease in LXR receptor activity in the presence of the test compound is indicative that the test compound is useful in promoting glial development.

0 In some embodiments, the LXR receptor and the test compound may be contacted in the presence of an LXR activator, such as an LXR ligand, for example an oxysterol, and the effect of the test compound on the binding or activation of the LXR receptor by the LXR activator determined.

.5

A test compound which promotes neurogenesis may induce, increase or enhance neuronal development, differentiation and/or maturation or the onset of a neuronal phenotype in a neural stem, embryonic stem, progenitor or precursor cell.

>0

In other embodiments, the LXR receptor and the test compound may be contacted in the presence of a LXR suppressor, for example an anti- LXR RNAi molecule, and the effect of the test compound determined.

15 A test compound which promotes gliogenesis may induce, increase or enhance glial development, differentiation and/or maturation or the onset of a glial phenotype in a neural stem, embryonic stem, progenitor or precursor cell.

30 LXR receptor activity may be determined by any convenient method. For example, observation can be made of differentiation of a stem, progenitor or neuronal precursor cell that expresses sox2, Lmxla, Lmxlb, msxl, otx2, pax2, fox2a, Raldhl or Aldehyde dehydrogenase type 2, wntl, wnt-5a, dkk2, RC2, nestin, neurogenin2, neurogeninl,

mashl, engrailed 1 or nurrl into neurons, in particular dopaminergic neurons by: the acquisition of marker expression such as pitx3, c- ret, tyrosine hydroxylase, dopamine transporter or GIRK channel; absence of Gamma-Amino-butyric acid (GABA) , Glutamic acid decarboxylase (GAD) , serotonin or dopamine beta-hydroxylase (DBH) ; morphological differentiation, as assessed by the extension of neurites (expressing MAP2) or axons (expressing neurofilament); increased survival of cells as measured by increased numbers of cells expressing any of the above mentioned markers; increased neurotransmitter (dopamine) release, as measured by HPLC; increased of electrophysiological properties characteristic of mature neurons; increased signalling as measured by increased tyrosine, serine or threonin phosphorylation as measured in protein gels or MALDI-TOF; changes in the cells as detected by Biacore; and/or increased transcription as measured by luciferase or GFP or other reporter systems for instance TOPPFLASH assay to report beta catenin transcription, or promoters involved in dopaminergic differentiation coupled to luciferase or GFP or other reporter systems, such as the genes expressed in progenitor cell ^' s.

Another aspect of the invention provides a method of obtaining a factor or factors which, either alone or in combination, promote neurogenesis, the method comprising:

(a) treating a neural stem progenitor or precursor cell as described herein, with a LXR receptor or LXR ligand in the presence and absence of one or more test compounds; and

(b) determining proliferation, self-renewal, survival and/or dopaminergic development, induction, differentiation, or maturation of the cell and comparing the extent of the proliferation, self- renewal, survival and/or dopaminergic development, induction, differentiation or maturation in the presence and absence of the one or more test compounds, whereby said factor or factors is obtained.

Thus, the method may screen for a compound which modulates the ability of a LXR receptor to induce proliferation, self renewal, dopaminergic development, differentiation, maturation and/or acquisition of a dopaminergic fate in stem, neural stem, precursor, 3 progenitor or neural cells.

Such a method may include:

(i) providing stem, embryonic stem, neural stem, progenitor, precursor or neural cells in the presence of a LXR activator and one

0 or more test compounds;

(ii) analysing the proportion of such cells which adopt a neuronal, preferably a dopaminergic, fate or phenotype and/or respond to the LXR activator; (iii) comparing the proportion of such cells which adopt a neuronal,

5 preferably a dopaminergic, fate with the number of such cells which adopt a neuronal, preferably a dopaminergic fate or phenotype and/or respond to the LXR activator in comparable reaction medium and conditions in the absence of the test compound or test compounds . A difference in the proportion of neurons between the treated and

0 untreated cells is indicative of a modulating effect of the relevant test compound or test compounds.

Neuronal cell numbers and/or differentiation may be increased relative to glial cell numbers and/or differentiation. !5

An LXR activator is a compound which increases the amount of LXR signalling in a cell. LXR activators are described in more detail above .

30 A cell may be analyzed for differentiation to a neuronal phenotype, preferably a dopaminergic phenotype, e.g. by detecting the expression of a marker or markers of the dopaminergic phenotype as discussed herein. Tyrosine hydroxylase (TH), DAT, AADC, GIRK2,

Nurrl, Pitx3, engrailed 1 or 2, Lmxlb, Lmxla, Raldhl, Foxa2, Msxl, Otx2, Pax2, Ngn2, sox2 are markers of the dopaminergic phenotype .

Another aspect of the invention provides a method of obtaining a factor or factors which, either alone or in combination, promote gliogenesis, the method comprising:

(a) treating a neural stem progenitor or precursor cell as described herein, with an LXR suppressor in the presence and absence of one or more test compounds; and (b) determining proliferation, self-renewal, survival and/or glial development, induction, differentiation, or maturation of the cell and comparing the extent of the proliferation, self-renewal, survival and/or glial development, induction, differentiation or maturation in the presence and absence of the one or more test compounds, whereby said factor or factors is obtained.

Thus, the method may screen for a compound which modulates the ability of an LXR suppressor to induce proliferation, self renewal, glial development, differentiation, maturation and/or acquisition of a glial fate in stem, neural stem, precursor, progenitor or neural cells .

A LXR suppressor is a compound which reduces the amount of LXR signalling in a cell. LXR suppressors are described in more detail above.

Such a method may include:

(i) providing stem, embryonic stem, neural stem, progenitor, precursor or neural cells in the presence of a LXR suppressor and one or more test compounds;

(ii) analysing the proportion of such cells which adopt a glial fate or phenotype and/or respond to the LXR suppressor; (iii) comparing the proportion of such' cells which adopt a glial fate with the number of such cells which adopt a glial fate or

phenotype and/or respond to the LXR suppressor in comparable reaction medium and conditions in the absence of the test compound or test compounds. A difference in the proportion of glial cells between the treated and untreated cells is indicative of a modulating effect of the relevant test compound or test compounds .

Glial cell numbers and/or differentiation may be increased relative to neuronal cell numbers and/or differentiation.

The cell may be treated with the LXR activator or suppressor by addition of the LXR activator or suppressor to in vitro culture containing the cell, or by introduction of the LXR activator or suppressor into the cell.

A screening method may employ an inducible promoter operably linked to nucleic acid encoding a test compound. Such a construct is incorporated into a host cell and one or more properties of that cell under the permissive and non-permissive conditions of the promoter are determined and compared. The property determined may be the ability of the host cell to induce a neuronal or glial phenotype in a stem, neural stem, precursor, progenitor or neural cell in the presence of LXR activator or suppressor. A difference in that ability of the host cell between the permissive and non- permissive conditions indicates that the test compound may be able, either alone or in combination, to enhance proliferation and/or self-renewal and/or induction of a neuronal or glial fate and/or neuronal or glial differentiation, survival or development in a stem, neural stem or progenitor or precursor cell or enhance neuronal or glial induction or differentiation in a neuronal cell in the, presence of LXR activator or suppressor, respectively.

The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using

routine skill and knowledge. The skilled person is aware of the need to employ appropriate control experiments.

Compounds which may be screened may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used.

Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate an interaction. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.

The amount of test substance or compound which may be employed in a screening method will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to ImM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to 100 μM, e.g. 0.1 to 50 μM, such as about 10 μM. Greater concentrations may be used when a peptide is the test substance. Even a molecule which has a weak effect may be a useful lead compound for further investigation and development .

The screening method may further comprise identifying the test compound as a modulator of LXR signalling receptor activity, which may be useful in promoting neurogenesis or gliogenesis. A test compound which increases LXR signalling receptor activity may be identified as useful in promoting neurogenesis and a test compound which reduces LXR signalling receptor activity may be identified as useful in promoting gliogenesis.

A method may further comprise purifying and/or isolating the identified test compound from a mixture or extract, i.e. reducing the content of at least one component of the mixture or extract, e.g. a component with which the test substance is naturally associated. The purifying and/or isolating may employ any method known to those skilled in the art. The method may include determining the ability of one or more fractions of a test mixture or extract to modulate the LXR signalling activity.

The test compound may be developed to obtain peptidyl or non- peptidyl mimetics, e.g. by methods well known to those skilled in the art and discussed herein.

The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying amino acid residues in the peptide, e.g. by substituting each residue in turn (for example with alanine i.e. 'alanine scanning ¹ ). These parts or residues constituting the active region of the compound are known as its "pharmacophore" .

Cholesterol derivatives may be employed as mimetics. Cholesterol derivatives may be produced, for example, by hydroxylation or the addition of acid groups.

Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. 5 Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

In a variant of the above approach, the three-dimensional structure .0 of a ligand and its binding partner (the LXR receptor) are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.

L5 Another aspect of the invention provides a compound identified by a screening method above or mimetic thereof, for use in a method of treating a neural cell and/or a method of treatment of a condition characterised by neuronal loss, damage or dysfunction, such as neurodegenerative diseases or disorders. A compound or mimetic may

20 be provided in an isolated and/or purified form, i.e. substantially pure. This may include being in a composition where it represents at least about 90% active ingredient, more preferably at least about 95%, more preferably at least about 98%. Such a composition is preferably a pharmaceutical composition and may include inert 5 carrier materials or other pharmaceutically and physiologically acceptable excipients . An active ingredient means a pharmaceutically active substance, as opposed to e.g. a buffer or carrier material included to stabilise the active ingredient or facilitate its administration. 0

A test compound or mimetic thereof may, as a further step, be formulated into a medicament and optionally used in a method of treatment as described herein.

Thus, the present invention extends in various aspects not only to a compound identified using the present methods in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a compound, a method comprising administration of such a composition to a patient, for instance in treatment (which may include preventative treatment) of neurodegenerative disease or neuronal loss, use of such a compound in manufacture of a composition for administration to such a patient, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients. Pharmaceutical compositions and therapeutic methods are part of the present invention and are described in more detail below.

Other aspects of the invention relate to the identification of genes which are involved in LXR signalling. A method of identifying a member or component of the LXR signalling pathway may comprise; determining the amount of expression of a population of nucleic acid molecules, proteins or lipids in test cells, wherein said test cells are selected from the group consisting of LXRcf, LXRβ ^" and LXRβ ^" /LXRcf cells and cells stimulated with LXR ligand, comparing the amount of expression of each member of said population of nucleic acid molecules, proteins or lipids in the test cells relative to control cells, and; identifying one or more members of said population of nucleic acid molecules, proteins or lipids whose expression is altered in said test cells relative to control cells, wherein said identified nucleic acid molecules, proteins or lipids are candidate members of the LXR signalling pathway.

A member of the population of nucleic acid molecules, proteins or lipids whose expression is reduced in LXRof, LXRβ ^" or LXRβ ^~ /LXRof cells may be a candidate member of the LXR signalling pathway.

A member of the population of nucleic acid molecules, proteins or lipids whose expression is increased in cells stimulated with LXR ligand, such as oxysterol, may be a candidate member of the LXR signalling pathway.

Suitable nucleic acid molecules include RNA or cDNA molecules. The population of nucleic acid molecules may comprise all the transcribed nucleic acid (i.e. mRNA or cDNA) in the cell) .

The amount of expression of a population of nucleic acid molecules in a cell may be determined by any convenient method, including northern blotting, PCR or gene array hybridisation. Protein and lipid expression can be analysed by immunohystochemistry, ELISA, enzyme-based assays, proteomic methods, mass spectrophotometry or arrays to analyze the entire proteome or "lipidome".

In some embodiments, population of nucleic acid molecules may be contacted with a nucleic acid array. A nucleic acid array comprises a population of nucleic acid sequences immobilised on a solid support .

Typically, a nucleic acid array may comprise nucleotide sequences. In some embodiments, one or more chromosomes of a cell may be represented by the DNA sequences on the array or the entire transcriptome may be represented.

A nucleic acid array comprises a population of nucleotide sequences immobilised on a solid support. The number of nucleotide sequences deposited on the array generally may vary upwards from a minimum of at least 10, 100, 1000, or 10,000 to between 10,000 and several

million depending on the technology employed. Nucleic acid arrays are well known in the art and may be produced in a number of ways. For example, the nucleotide sequence may be synthesized ex situ using an oligonucleotide synthesis device, and subsequently deposited using a microarraying apparatus or synthesized in situ on the microarray using a method such as piezoelectric deposition of nucleotides .

Each nucleotide sequence in the population is located in a particular defined position on the support and hybridises a nucleic acid which is transcribed by the cell. The amount of hybridisation of transcribed nucleic acid at each position is indicative of the amount of expression of that transcribed nucleic acid by the cell.

Prior to hybridisation with the sequences of an array, the amplified nucleic acid molecules may be labelled. Labelling of amplification products may be achieved by standard methods. For example, products may be amplified (linearly or exponentially) from an amplification product using synthetically labelled oligonucleotides (e.g. containing Cy5- or Cy3-modified nucleotides or amino allyl modified nucleotides, which allow for chemical coupling of the dye molecules post amplification) , or modified or labelled nucleotides during the amplification reaction. Suitable labels include fluorescent labels, such as cyanine 3 or cyanine 5. The labelled extension products may then be hybridised to an array using standard techniques.

The nucleic acid sequences on the array to which the product hybridises may be determined, for example by measuring and recording the label intensity at each position in the array, for example, using an automated DNA microarray reader.

The presence or amount of hybridisation of transcribed nucleic acid molecules to a nucleotide sequence displayed in a region of the

array may be indicative of the amount of expression of that nucleic acid within the cell.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety.

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

Figure 1 shows that Liver X receptors are expressed during dopaminergic neurogenesis. (A-B) Quantitative PCR analysis revealed that LXRα and LXRβ expression increases in both the dorsal and ventral midbrain during DA neurogenesis (boxes) .

Figure 2 shows that LXR receptors are transcriptionally active in ventral midbrain-derived SN4741 cells by analysis of expression of LXRα, LXRβ, and RXRα transcripts in total RNAs from dopaminergic (SN4741, MN9D) and non-dopaminergic (HEK-293) cells analyzed by RT- PCR. The dopaminergic marker Nurrl was used as a positive control and Alkl, a known LXR interacting protein, was also detected. GAPDH was used as the internal control.

Figure 3 shows that LXR receptors are transcriptionally active in ventral midbrain-derived SN4741 cells by analysis of luciferase activity in SN4741 cell extracts transfected with a luciferase reporter plasmid containing three LXR binding sites (LXRE-Luc) and 200ng of the nuclear receptors, LXRα and RXRα (B) and LXRβ and RXRα (C) , as indicated. Cells were stimulated for 24h with an LXR agonist, 22 (R) -hydroxycholesterol (22-HC; lOμM) , or an RXR agonist,

9cis Retinoic Acid (9cRA; 10μM) , or both ligands simultaneously (9cRA+ 22-HC) . Firefly luciferase activity was normalized to Renilla luciferase activity, and the values are expressed as fold activation over the normalized basal LXRE-Luc activity set to 1. Data are the means ± S.E. (bars) of a representative experiment (n=3) , and the experiments were performed five times with similar results .

Figure 4 shows that 22-hydroxycholesterol promotes neurogenesis in mouse ventral midbrain DA progenitors in vitro. Ell mouse cortical and ventral midbrain primary cultures were treated with vehicle or lOμM 22-hydroxycholesterol and differentiated for 3DIV. Immunostaining with an antibody against RC2 showed a reduction in the percent of RC2+ radial glia following treatment with 22-HC in ventral midbrain primary cultures but not in cortical progenitors (A-D) . The proportion of cells incorporating Brdϋ, a proliferation marker, also decreased in ventral midbrain primary cells treated with LXR ligand (E-H) . Treatment with lOμM 22-HC enhanced the number of TH+ DA neurons in ventral midbrain, but not cortical cultures, after 3 days in vitro (I-L) .

Figure 5 shows quantitative analysis of immunostaining of mouse ventral midbrain DA progenitors in vitro. M shows a reduction in the percent of RC2+ radial glia following treatment with 22-HC in ventral midbrain primary cultures (M) but not in cortical progenitors. N shows that proportion of cells incorporating BrdU, a proliferation marker, also decreased in ventral midbrain primary cells treated with LXR ligand. O shows that treatment with lOμM 22- HC enhanced the number of TH+ DA neurons in ventral midbrain, but not cortical cultures, after 3 days in vitro. Data represent the results of four independent experiments performed in duplicate. Statistical analysis was performed using two-tailed unpaired t- tests. Significance levels were *P<0.05; **0.01<P<0.001; ***P<0.001.

Figure 6 shows the increase in proliferating progenitors in LXR null mice. Quantitative PCR analysis showed that loss of LXR function in the VM resulted in no changes in the prototypical dopaminergic patterning markers Shh (A), Fgf8 (B), and 0tx2 (C) mRNAs .

Figure 7 shows that LXR null mice exhibited no changes in the number of Sox2+ precursors in the ventral midbrain.

Figure 8 shows immunohistochemistry performed on coronal sections through the ventral midbrain of LXR null mice using anti-phospho- histone H3 antibody. This revealed significant increases in the number of proliferating progenitors residing in the ventricular zone of the LXR null midbrains.

Figure 9 shows that loss of both LXRα and LXRβ resulted in increases in the number of proliferating progenitors residing in the ventricular zone of the LXR null midbrains (F) and the number of proliferating dopaminergic precursors incorporating BrdU during S phase (G) . Data represent the quantitative analysis of serial sections through the entire midbrains of five embryos from each genotype. Statistical analysis was performed using one-way ANOVA (compared with wildtype) , with Fisher's post hoc test. Significance levels were *P<0.05; **0. OKKO.001.

Figure 10 shows immunohistochemistry performed on coronal sections through the ventral midbrains of Ell wildtype and LXR knockout mice. No changes were observed in the number of RC2+ radial glia in the midbrains of LXR null mice (A, D, G, J) . A significant loss in the number of TH+ dopaminergic neurons was found in LXRα null mice, while LXRβ and LXRαβ null mice displayed losses of almost

70% (B, E, H, K) . All LXR null genotypes displayed a 50-60% loss in the number of immature Tujl+ neurons (C, F, I, L).

Figure 11 shows quantitative analysis of neurogenic defects and impaired neurogenic networks in LXR null mice. No changes were observed in the number of RC2+ radial glia in the midbrains of LXR null mice. (M) A significant loss in the number of TH+ dopaminergic neurons was found in LXRα null mice, while LXRβ and LXRαβ null mice displayed losses of almost 70%. (N) All LXR null genotypes displayed a 50-60% loss in the number of immature Tujl+ neurons (O) . Quantitative analysis of the number of cleaved caspase- 3+ cells revealed no changes in the level of apoptosis in any of the LXR genotypes compared to wildtype mice (P) .

Figure 12 shows quantitative analysis of markers involved in dopaminergic maturation. (Q) Nurrl and (R) Pitx3 mRNAs were downregulated in all LXR null mice compared to wildtype animals. Key determinants of dopaminergic lineage specification such as (S) Lmxla and (T) Msxl were unaffected. (U, V) Lmxlb and Adh2 transcripts, which mark cells with radial glia morphology were upregulated in LXR null genotypes . Data represent the quantitative analysis of serial sections through the entire midbrains of four Ell embryos from each genotype. Statistical analysis was performed using one-way ANOVA (compared with wildtype), by with Fisher's post hoc test. Significance levels were *P<0.05; **0. OKKO .001 and ***P<0.001.

Figure 13 shows immunohistochemical analysis of coronal sections through the ventral midbrains of E13.5 wildtype and LXR null mice which indicate that the loss of midbrain dopaminergic neurons in LXR null mice is accompanied by enhanced gliogenesis in E13.5 LXR null mice. A progressive loss of TH+ dopaminergic neurons is found in LXR null mice with LXRβ-/- and LXRαβ-/- mice displaying the most severe losses (A, D, G, J) . LXR null mice also exhibited a 2-4-fold increase in the number of RC2+ radial glia processes throughout the VM (B, E, H, K, N). No changes in the total number of cells in any of the isoform knockouts was observed (C, F, I, L).

Figure 14 shows quantitative immunocytochemical analysis of serial sections through the entire midbrains of six E13.5 embryos from each genotype. Statistical analysis was performed using one-way ANOVA (compared with wildtype) , by with Fisher's post hoc test. Significance levels were *P<0.05; **0. OKKO .001 and ***P<0.001.

Figure 15 shows coronal sections through the midbrains of Pl wildtype and LXRcφ-/- mice. At Pl, LXRαβ-/- mice retain their dopaminergic deficits (TH) (A, B) and exhibit increased numbers of GFAP+ astrocytes throughout the ventral midbrain (C-F) .

Figure 16 shows the results of quantitative PCR on the VMs of wildtype and LXR null mice. General markers of the neurogenic capacity of the ventral midbrain including Wnt-1 (G) Neurogenin-2 (H) and the proneural genes Hes5 (T) and Dill (J) were downregulated in virtually all the LXR isoform knockouts. Additionally, Tis21, a marker of neurogenic asymmetric division was also significantly reduced in the VM of LXRαβ nulls (K) . Precocious expression of the astrocytic marker GFAP was observed, particularly in LXRβ and LXRαβ null mice (L) . Statistical analysis was performed using one-way ANOVA (compared with wildtype), by with Fisher's post hoc test. Significance levels were *P<0.05 and **0.01<P<0.001.

Figure 17 shows ventral midbrain dopaminergic neurons treated with oxysterols fail to develop in the absence of intracellular LXR.

Tyrosine hydroxylase (TH) immunostaining shows a two-fold increase in the number of positive cells out of the total number of cells (Hoechst 33258) three days after treatment with 22- hydroxycholesterol in wildtype ventral midbrain progenitor cultures (A, C) while ventral midbrain progenitors isolated from LXRotβ-/- mice failed to differentiate in the presence of oxysterol compared to vehicle treatment (B, D) .

Figure 18- shows a quantitative analysis of the data shown in figure 17. Data represent the results of 3 independent experiments performed in duplicate. Statistical analysis was performed using two-tailed unpaired t-tests. Significance levels were *P<0.05; 5 **0.0KP<0.001; ***P<0.001.

Figure 19 shows a schematic representation of the sequential steps designed to induce a dopamine neuronal phenotype from undifferentiated hES cells. hES cells are first plated on stromal

LO feeder cells (at low density) in medium SRM. From day 12 to 16 SHH and FGF8 are added to SRM. From day 16 to the end of the differentiation N2 medium with BDNF and AA is used, supplemented with other factors (FGF8, SHH) at the adequate time points. At the rosette stage, these neuroepithelial structures are replated on

L5 coated dishes in a feeder-free system for differentiation (passage

1, Pl) . Treatment with the oxysterol 22-R-hydroxycholesterol started at passage 2 (P2) , until the end of the differentiation.

Figure 20 shows oxysterols increase dopaminergic neuron

20 differentiation from hES cells. hES cells (line H9) on passage 2 of dopaminergic differentiation were treated with different concentrations of 22-R-hydroxycholesterol (Control, 0,lμM, 0,5 μM, lμM and 5 μM) . A) Double ICC for TH and TuJl in the different treatment groups; nuclei appear counter-stained with Hoechst. B, C) 25 Represent the percentages of TuJl-positive and TH-positive neurons among all cells. D) Percentage of TH-positive cells among all TuJl- positive neurons. (*) p < 0.05; (**) p < 0.01.

Figure 21 shows oxysterols increase dopaminergic neuron

30 differentiation from hES cells. hES cells (line HS181) on passage 2 of dopaminergic differentiation were treated with different concentrations of 22-R-hydroxycholesterol (Control, 0,lμM, iμM and 5 μM) . A) Double ICC for TH (red) and TuJl (green) in the different treatment groups; nuclei appear counter-stained with Hoechst. B, C)

Represent the percentages of TH-positive (B) and TuJl-positive neurons (C) among all cells. The percentages of TuJl- and mainly TH- positive neurons were significantly increased by oxysterol treatment. However, higher concentrations (5 μM) were toxic for the cells. D) Percentage of TH-positive cells among all TuJl-positive neurons. (*) p < 0.05; (**) p < 0.01.

Figure 22 shows oxysterols increase dopaminergic neuron differentiation from mES cells. Control- or LXRBeta-overexpressing Rl mES. Differentiating mES (on day 8) were treated with/without the oxysterol 22-R-hydroxycholesterol at 0,5 μM until de end of the differentiation. A) Double ICC for TH and TuJl in the different treatment groups; nuclei appear counter-stained with Hoechst in blue. B, C) Represent the percentages of TH-positive (B) and TuJl- positive colonies (C) among all colonies. D) Represent the number of TH+ cells per area Unit (6250 μm2) . (*) p < 0.05; (**) p < 0.01.

Experiments Materials and Methods Cell Cultures hES cell lines H9 (WA-09, XX, passages 35-40) and HS181 (XX, passages 35-45) were cultured on mitotically inactivated human foreskin fibroblasts (hFF; ATCC; CRL-2429) , in a medium containing: KO- DMEM medium (Invitrogen) , 20% Knockout serum replacement (SRM; Invitrogen) , 2mM L-glutamine (Invitrogen) , 1% nonessential amino acids (Invitrogen), O.lmM beta-mercaptoethanol (Sigma), 4 ng/ml of basic fibroblast growth factor (bFGF) (R&D Systems) and Pen./Strept. (10.000 U/ml; Invitrogen). Medium was changed daily.

mES (Rl) were cultured on gelatinized plates in a medium containing: KO- DMEM medium (Invitrogen) , 15% Knockout serum replacement (SRM; Invitrogen) , 2mM L-glutamine (Invitrogen) , 1% nonessential amino acids (Invitrogen), O.lmM beta-mercaptoethanol (Sigma), LIF 1000 U/ml (ESGRO, Chemicon) and Pen./Strept. (10.000

ϋ/ml; Invitrogen) . Proliferating cells were nucleofected with expression vectors (pCMV-hLXRBeta/FLAG-Ires-Neo or pCMV-Empty/FLAG- Ires-Neo, as a control) . according to protocol (mouse ES nucleoporator kit, AMAXA) , replated on gelatin coated-plates in mES medium. Neomycin (Geneticin, G-418, Invitrogen) selection (300 μg/ml) started 48 h post-nucleofection.

The hFF were propagated in DMEM medium containing 10% of fetal bovine serum (FBS; Invitrogen) .

Stromal PA6 cells were maintained in alpha-minimum essential medium (Invitrogen) containing 10% FBS and 2 πiM L-glutamine. hFF and PA6 cells were mitotically inactivated before use, by treatment with mitomycin C (lμg/ml; Roche) for 2-4 h at 37°C. All cell lines were maintained at 37°C, 5% CO ₂ and 95% humidity.

Transient transfection studies were performed in SN4741 cells, grown at 37 ⁰ C as previously published. Cells were plated in 24-well plates (5x105 cells/well) 24 h before transfection and transfected with 1. Oμg of plasmid DNA/well complexed with 2μl of Lipofectamine 2000 (Invitrogen) . Typically, cells were transfected with 400 ng of the reporter construct and 200 ng of receptor expression vectors. A reporter gene expressing the Renilla luciferase (pRL-TK, Promega) was cotransfected (10 ng) in all experiments as an internal control for normalization of transfection efficiency. After a 5-h incubation, the lipid/DNA mix was replaced with fresh 2.5% serum medium containing vehicle or appropriate ligand (lOμM), as specified in the figure legend. Luciferase activities were assayed 24 h later using Dual- Luciferase Reporter Assay System (Promega) , following the manufacturer's protocol.

Neural induction and differentiation

Neural differentiation of hES cells was induced by means of co- culture on PA6 stromal cells, according to Perrier et al., 2004. hES were plated at 20 X 10 ³ cells on a confluent layer of PA6 cells

(mitotically inactivated) in 6-cm cell culture plates in serum replacement medium (SRM) containing KO-DMEM, 15% Knockout serum replacement, 2 rtiM L-glutamine and 0. ImM beta-mercaptoethanol . At day 12 SHH (200 ng/ml; R&D Systems) and FGF8 (25-100 ng/ml; R&D Systems) were added to the medium. After 16 days cultures were switched to N2 medium in the presence of SHH, FGF8, brain-derived neurotrophic factor (BDNF; 20 ng/ml; R&D Systems) and ascorbic acid (AA, 0.2 mM; Sigma) .

At day 20 SHH and FGF8 were withdrawn, following the differentiation until day 25-28. Rosettes structures containing neural precursors were manually isolated and replated on polyornithine (15 μg/ml; Sigma) /Laminin (1 μg/ml) -coated culture dishes in N2 medium supplemented with SHH, FGF8, AA and BDNF (Passage 1, Pl) . After 1 week, cells were mechanically passaged after exposure to Ca ² /Mg ² -free Hanks balanced salt solution (HBSS; Invitrogen) for 1 h at room temperature and spun at 200 X g for 5 min (Passage 2, P2) . Cells were resuspended in N2 medium, replated again onto polyornithine/Laminin-coated culture dishes (50-100 x 10 ³ cells per cm2) in the presence of SHH, FGF8, AA , BDNF and different concentrations of oxysterols (22-R-hydroxycholesterol; Sigma) . After 7 days cells were differentiated in the absence of SHH and FGF8 but in the presence of BDNF, glial cell line-derived neurotrophic factor (GDNF 10 ng/ml) , transforming growth factor type B3, dibytyril cAMP, AA, and different concentrations of oxysterols for 1 more week. See schematic representation in Figure 1.

Neural differentiation of mES cells was induced by co-culture on PA6 stromal cells, in accordance with the protocol by Barberi et al . , 2003. mES were plated at low density (150 cells/cm2) on a confluent layer of PA6 cells (mitotically inactivated) in 24 well- plates in serum replacement medium (SRM) containing KO-DMEM, 15% Knockout serum replacement, 2 mM L-glutamine and 0. ImM beta- mercaptoethanol. At day 5, SHH (200 ng/ml; R&D Systems) and FGF8 (25-100 ng/ml; R&D Systems) were added to the medium. After 8 days

cultures were switched to N2 medium in the presence of SHH, FGF8 and FGF2 (10 ng/ml) . At day 11 SHH and FGF8 were withdrawn, following the differentiation until day 14 in medium N2 containing AA (0.2 mM; Sigma), BDNF (20 ng/ml) and GDNF (lOng/ml) . Treatments with or without 22-R-hydroxycholesterol (0.5μM) started on day 8 until the end of differentiation.

Primary ventral mesencephalic and cortical cultures and immunocytochemistry Experiments using Ell c57/B6 mice or LXRab-/- mice were performed in accordance and approval of the local ethical committee (Stockholms Norra Djurforsόketiska Namnd) . Ventral mesencephala or cortices were manually dissected and plated at a final density of 125, 000 cells/well on poly-D-lysine-coated 48-well plates. Primary cultures were grown in serum-free N2 media consisting of 1:1 mixture of F12 and DMEM with 10 ng/ml insulin, 100 μg/mL apo-transferrin, 100 μM putrescine, 20 nM progesterone, 30 nM selenium, 6 mg/ml glucose, and 1 mg/ml BSA.

Cells were treated with lOμM of 22-hydroxycholesterol or GW3965 or vehicle for 3 days in vitro, fixed for 15 minutes with 4% paraformaldehyde, washed in PBS, and used for immunocytochemical analysis. Cultures were blocked for 1 hour at room temperature in PBST (Ix PBS, 1% BSA, and 0.3% Triton X-100) and incubated overnight at 4 ⁰ C with the corresponding primary antibody diluted in PBST. The following antibodies were used: rabbit anti-tyrosine hydroxylase (TH), 1:1000 (Pel-freeze); mouse anti-RC2 1:500 (DSHB), rat anti- BrdU 1:200 (Abeam). After washing, cultures were incubated with 1:250 dilutions of Cy2- or Rhodamine-coupled secondary antibodies (Jackson ImmunoResearch) in PBST. Cultures were then rinsed twice in PBS. For BrdU analysis, pulsed cell's were treated for 30 minutes with 2N HCl and then processed for secondary antibody staining, as indicated above. Hoescht staining was performed by permeablizing cells with a 0.3% Triton-X 100/PBS solution for 5 minutes followed

by incubation with Hoescht 33258 (Sigma) for 10 minutes. Cells were counted and images were taken with a with a Zeiss Axioplan IOOM microscope (LD Achrostigmat 2Ox, 0.3 PHl 0-2 and LD Achroplan 4Ox, 0.60 Korr PH2 0-2) and collected with a Hamamatsu camera C4742-95 (with OpenLab software) .

Cells were fixed in 4% paraformaldehyde and stained with rabbit polyclonal anti TH (1:500; Pel-Freez Biologicals) and mouse monoclonal anti B-tubulin III (TuJl; 1:1000; Promega) . Donkey anti- rabbit cy3- and goat anti mouse cy2-labeled (Jackson Immunoresearch) were used as secondary antibodies. Cell nuclei were visualized by Hoechst 33258 counter-staining.

CeU counts and Statistical Analyses Percentage of TH- or TuJl-positive neurons were obtained from 4 different experiments for both human cell lines (H9 and HS181); 10 to 15 random fields per well were analyzed. Percentages of TH- or TuJl-positive neurons were compared with ANOVA and Dunnett's Multiple Comparison post hoc analysis (GraphPad Prism) . All data are expressed as mean ± SEM. For mES cells, we analyzed the percentages of TuJl+ or TH+ colonies related to total colonies, and the number of TH+ cells per area Unit (6250 μm2) . Data from different groups were compared with ANOVA and Dunnett's Multiple Comparison post hoc analysis .

Real-time RT-PCR

RNA from Ell ventral mesencephala of wildtype or LXR null mice,

SN4741, MN9D, or HEK293 cells was extracted using RNeasy Mini Kit (Qiagen) . 1 μg of total RNA was treated with RQl RNase-free DNase (Promega, Madison, WI) and RNA integrity was assessed by electrophoresis. Briefly, 1 μg of RNA was reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen) and random primers (Invitrogen) (RT+ reaction) , and parallel reactions without reverse transcriptase enzyme were done as a control (RT- reaction) . RT-PCR

and quantitative PCR was performed as previously described. Primers were designed using Primer Express and sequences are available upon request.

LXR null mice and Immunohistochemistry

LXR null mice were previously described. Appropriate time-mated embryos were fixed in ice cold 4% PFA for 6 hours, cryoprotected in 20% sucrose, and frozen in OCT compound at -70 ⁰ C. Next, serial coronal 14 μM sections of the entire ventral midbrain were cut on a cryostat.

Immunohistochemistry was performed on 4% paraformaldehyde-postfixed slides. Incubations were carried out at 4°C overnight with the following antibodies diluted in PBS, pH 7.4, 1% BSA, and 0.3% Triton X-100 solution: rabbit anti-Nurrl (1:200 dilution, Santa Cruz Biotech.), rabbit anti-TH (1:500, Pel-Freeze), rat anti-BrdU (1:200, Abeam), mouse anti-phosphorylated histone H3 (SerlO) (1:100, Upstate), mouse anti-RC2 (1:200, DSHB), guinea pig antiglutamate transporter, GLAST (1:2000, Chemicon) , mouse anti-Tujl (1:1000 Promega) , rabbit anti-GFAP (1:1000 Dako) , rabbit anti-Sox2 (1:250, gift from T. Edlund) and rabbit anticleaved caspase-3 (Aspl75)

(1:100, Cell Signaling Technology). After washes, the sections were blocked for 30 minutes in dilution buffer and then incubated for 2-4 hours with a secondary antibody Cy2/3 (cyanine) -coupled donkey anti- mouse IgG, Cy2-coupled donkey anti-rabbit IgG (1:200 dilution, Jackson ImmunoResearch) . Immunostaining was visualized by using a Zeiss HBOlOO microscope. For BrdU analysis, pregnant mice were injected with 0.5 ml of a 1 mg/ml Brdϋ/saline solution 6 hours before sacrifice and then sectioned. Pretreatment of 14μM histological sections with 2N HCl for 15 minutes prior to pre- incubation with primary antibody was needed for detection of BrdU.

Stainings were visualized using a Zeiss HBOlOO microscope and images were collected with a Hamamatsu camera C4742-95 and processed with OpenLab software.

Results

LXRs are spatially and temporally expressed in proliferating progenitors of the developing ventral midbrain

The birth of DA neurons in the ventral midbrain starts between embryonic day 10.5-11.5 (ElO .5-EIl .5) in mice. To identify whether LXRs are involved in this process, we first examined the temporal and spatial expression patterns of LXRα and LXRβ in the developing mouse mesencephalon by quantitative PCR. Transcripts of both LXRα and LXRβ were detected as early as E9.5 in the dorsal and ventral mesencephalon throughout the DA neurogenic period (Figure 1 A and

B). Notably, LXR mRNAs were upregulated between ElO.5 and Ell.5, at the onset of DA neurogenesis suggesting a role for these receptors in the generation of DA neurons.

Oxysterols activate transcription in dopaminergic cells and enhance the differentiation of ventral midbrain progenitors.

Next, we sought to evaluate the responsiveness of dopaminergic cells to LXR ligands. To address this, we screened two dopaminergic cell lines: MN9D and SN4741 cells and nonneuronal HEK293 cells. Of these three cell lines, only SN4741 dopaminergic cells expressed detectable levels of both LXRα and LXRβ mRNAs and other nuclear receptors previously identified in dopaminergic neurons (Figure 2) . Additionally, SN4741 cells also expressed ALKl, a type I member of the TGF-β receptor family that has previously been shown to interact with LXR (Figure 2) .

To assay the transcriptional activity of both LXRs in dopaminergic cells, we examined the effect of 22-hydroxycholesterol alone, or in combination with 9-cis retinoic acid on the induction of a luciferase reporter gene driven by an LXREregulated promoter. LXRE- driven reporter activity was detected in SN4741 dopaminergic cells treated with oxysterols at concentrations ranging from l-10μM.

22-hydroxycholesterol treatment of dopaminergic cells transfected with LXRα or LXRβ resulted in a 2-fold increase in luciferase activity (Figure 3 B, C) . When dopaminergic cells were co- transfected with LXRα or LXRβ and treated with both oxysterols and retinoid ligands, these effects were potentiated. Moreover, co- transfection of LXRβ and RXRα in the presence of both ligands, induced a synergistic activation of LXRE-driven reporter activity by approximately 8-fold (Figure 3C) . These data identify the SN4741 cell line as a suitable cell-based system to assess LXR signals and show for the first time, that dopaminergic cells transcriptionally respond to oxysterols.

We next examined the influence of LXR activation on the differentiation of neural progenitors. To address this, we treated Ell primary cortical and ventral mesencephalic cultures with the LXR ligand 22-hydroxycholesterol. These experiments excluded treatment together with 9-cis RA so that activation of additional pathways, including Nurrl/RXRcx signals, would not mask the readout of any oxysterol-mediated effects. Interestingly, treatment of cortical progenitors resulted in no changes in the number of radial glia

(Figure 4A,B, Figure 5M), BrdU+ cells, (Figure 4E,F, Figure 5N) or TH+ DA neurons (Figure 4 I, J, Figure 50). In contrast, treatment of VM progenitors with 22-hydroxycholesterol (lOμM) resulted in an almost 2-fold increase in the number of TH+ DA neurons generated in VM progenitor cultures, providing indication of region-specific oxysterol effects (Figure 4K, L, Figure 50). Similar results were obtained using the synthetic LXR ligand GW3965. Additionally, we observed a modest decrease in the number of RC2+ radial glia (Figure 4G, H, Figure 5M) that was accompanied by a 30% decrease in the number of BrdU+ cells (Figure 4G, H, Figure 5N) . These data indicate that LXR agonists regulate DA neurons and radial glia in an opposing manner (increasing the number of DA neurons and decreasing number and proliferation of radial glia.

Increased progenitor proliferation in the VM of LXR null mice Having established that LXRs are expressed and developmentally regulated in the early VM and that dopaminergic cells respond to oxysterols, we examined the phenotypic consequences of diminished LXR function on dopaminergic development, using LXRα-/-, LXRβ-/-, and LXRαβ-/- embryos. In contrast to adult animals. Ell LXR null embryos exhibited no obvious gross anatomical defects in overall brain architecture, and more specifically in midbrain structure.

We examined the expression of transcription and secreted factors that regulate the expression Wntl, a factor that regulates several aspects of DA neuron development. While no change in the expression of otx2 and fgfβ mRNA was detected in the VM floor plate at Ell.5, we found a severe down regulation of Lmxlb mRNA as assessed by ISH. This reduction was more severe in the marginal zone, which was also reduced in size and cell numbers. Interestingly, despite the normal expression levels of otx2 and fgf8, wntl expression levels were reduced by 50% and pitκ3, a downstream gene regulated by Wntl and Lmxlb was also down regulated. In contrast, analysis of the Shh- Lmxla-Msxl pathway showed no change in mRNA expression levels in the lxr null embryos. We also examined whether the patterning of the ventral midbrain was altered in the LXR null embryos, but found no alteration in the expression of genes important for this process such as Shh, Fgf8, and Otx2 (Figure 6 A-C) . Histological analysis only revealed a slight redistribution of Lmxlaí cells, which instead of a predominantly lateral distribution in the floor plate, became more homogeneous. These results provide indication that the specification of DA neurons is not affected and patterning is minimally affected, despite the defect in the Lmxlb-Wntl-Pitx3 pathway. However the reduction of cells in the marginal zone, in the area were post mitotic DA cells appear, together with the previous

in vitro results, provided indication that there might be an alteration in cell cycle of DA progenitors.

We next investigated the proliferative potential of VM dopaminergic 5 progenitors in vivo, in the neuroepithelium of LXR knockout mice. Phospho-histone H3 labeling revealed proliferating progenitors in M phase of the cell cycle that were positioned in the apical portion of the neuroepithelium, in contact with the ventricular cavity. The number of positive cells increased by 22% and 31% in LXRα-/- and

LO LXRβ-/- mice, respectively (Figure 8, Figure 9F) . This increase was even greater in LXRαβ-/- embryos, which displayed a 45% increase in the number of phospho-histone H3+ cells compared to wild-type controls (Figure 9F) . The increase in proliferation was also confirmed by Brdϋ pulses, which revealed a 45% increase in the

L5 ^' number of cells that incorporate Brdϋ (57.7 ± 6.9 in wild-type and 83.7 ± 4.7 in LXRαβ-/- mice, Figure 9G). However, despite the increase in the number of proliferating progenitors in LXRαβ-/- mice, which extended more ventrally (Figure 8) , we did not detect any accumulation of cells in the ventricular zone, as we previously

20 reported in Ngn2 null mice.

We next examined whether increases in proliferation results from a deficient cell cycle exit of floor plate progenitors. Pulse chase experiments with BrdU followed by Ki67 staining after 24 hours 5 revealed that 50% of the cells in the floor plate of control mice and 20 % of the cells in the Ixrαβ-/- mice exit the cycle. Thus, our results indicate that the labelled DA progenitors cannot exit the cell cycle and became KiI67 negative and continue proliferative divisions, instead of starting neurogenic divisions.

30

These results confirmed our gain of function experiments in vitro and provided indication that LXRs negatively regulate progenitor proliferation in the ventral midbrain.

Dopaminergic neurogenesis is compromised in LXR null mice Further examination of Ell LXRα-/- mice showed a 33% loss in TH+ DA neurons in the subjacent marginal zone (Figure 1OB, E, and Figure UN) . The generation of dopaminergic neurons was more severely compromised in LXRβ-/~ and LXRαβ-/- mice, which displayed losses of almost 70% (Figure 1OB, H, K, and Figure UN) . To investigate whether the lack of dopaminergic neurons in LXR null mutants reflected a general reduction in the number of neurons, immunohistochemical analysis was performed with the general neuronal marker Tujl. All LXR null genotypes exhibited reductions in the number of Tujl-immunoreactive neurons present in the VM, with LXRβ- /-, and LXRαβ-/- mice being most affected (Figure IOC, I, F, L, and Figure 110) . Consistent with our observations of fewer DA cells in LXR null mice, we also observed decreases in Nurrl and Pitx3 expression (Figure 12Q, and R) . However, at Ell, none of the LXR knockouts showed any change in the number of RC2+ radial glia cells (Figure 1OA, D, G, J, and Figure HM) or in the transcript levels of two homeodomain proteins involved in establishing and maintaining dopaminergic progenitor identity, Lmxla and Msxl (Figure 12S, and T) .

Additionally, no significant increase in cell death was observed in the VM as assessed by the presence of active caspase-3 or pyknotic nuclei (Figure IIP) , providing indication that the DA deficits associated with ablation of LXRα or LXRβ represent true neurogenic deficits. However, we also detected an increase in Lmxlb and Raldhlal transcripts in the VM of all LXR null mice (Figure 12U, and V) , which together with an increased number of proliferating cells in the neuroepithelium provided indication that further phenotypic alterations may be underway and prompted us to examine later stages of development in LXR null mice.

XrXRs coordinate neurogenic and gliogenic networks in the developing VM

We next examined neuron and glial production in LXR null embryos at E13.5, a time period that marks the end of dopaminergic neurogenesis and beginning of astrogliogenesis . At E13.5, LXR null mice recovered some of the dopaminergic deficits that were detected earlier at Ell.5 (Figure 14M). However, at E13.5, LXRα-/- mice had approximately 30% loss of TH+ dopaminergic neurons compared to wild- type controls, while LXRβ-/- and LXRαβ-/- mice exhibited a more severe phenotype and showed about 30% less DA neurons compared to wildtype mice (Figure 13A,G, and J) . The overproduction of NE cells and decreased neurogenesis at Ell.5 in the absence of cell death led us to hypothesize that additional cell types were being generated, and prompted us to examine whether glial cell types were being produced at E13.5. Consistent with this possibility was the observation of an almost 4-fold increase in RC2+ radial glia cells in LXRαβ-/- mice at this stage (Figure 13K, and Figure 14N) . Thus, combined our observations provide indication that loss of LXR function favors VM progenitors acquiring a glial cell fate at the expense of DA neurogenesis.

To examine whether loss of LXR function resulted in permanent alterations in the VM, we examined the number of TH+ DA neurons and GFAP+ astrocytes at Pl. Indeed by Pl, LXRαβ-/- mice continued to exhibit markedly decreased numbers of DA neurons while displaying increased numbers of GFAP+ astrocytes (Figure 15A-F) . In line with the gliogenic phenotype observed at E13.5 and Pl, we observed a premature acquisition of the astrocytic marker GFAP at Ell.5 (Figure 16L) . Moreover, we also found that transcript levels of Ngn2, a proneural basic-helix-loop-helix (bHLH) gene required for dopaminergic neurogenesis 11, 12 were reduced in all LXR null genotypes (Figure 16H) . Instead, no changes were observed in any of the LXR null animals analyzed for Mashl, another bHLH proneural gene that is not required for DA neurogenesis. The decrease in Ngn2 mRNA

correlated well with the large decreases in the prototypical DA genes tyrosine hydroxylase and Nurrl.

We examined the expression of tis21 an anti-proliferative gene expressed by cells in the neuroepithelium as they undergo asymmetric neurogenic divisions, and in agreement with the decrease in progenitor cell cycle exit, we found a decrease in the expression of tis21 in the 2xrαβ ^";" mice (Figure 16K) . We then examined the expression of proneural genes involved in the initiation of neurogenesis, including dill, hes5 and ngn2. We found a downregulation of dill in the Ixrαβ ^"7' mice, and a very clear down regulation of hes5 and ngn2 in the three different Ixr knockouts (Figure 16G, I, and J) . Histological analysis showed a reduction in ngn2 mRNA in the VM, which was more pronounced in the floorplate compared to the adjacent basal plate.

The defect in Ngn2 expression resulted in loss of Tujl+ neurons that affected most ventral structures but predominantly affected DA neurons in the floor plate, which were absent in LXRcφ-/- mice, and severely reduced in LXRα-/- and LXRβ-/- mice. Motor-neurons were also found to be affected to a lesser extent. Other neuronal cell types are therefore also regulated and this indicates that LXRs regulate the acquisition of a pan-neuronal phenotype.

These findings show that LXRs may regulate different aspects of neurogenesis and prompted us to examine whether DA neurogenesis was impaired in Ixr null mice in vivo. To examine whether neurons were being born in the VM, we performed Tujl and PI double stainings to distinguish neuronal cell bodies from fibers crossing the midline. In control condition (wt) , abundant neurons were found both in the marginal zone of the floor plate, both in the midline compartment, but also laterally and in the basal plate. In lxrcf' ^~ and lxr$ ^~ ' ^~ mice, very few neurons were observed in the marginal zone of the floor plate (ixroT' ^' ) or emerging from it (Ixrβ ^" '' ^" ) . Unlike in control

brains, TuJ+ neurites crossing the midline followed a straight line, reflecting the presence of fewer cell bodies. Finally, nearly no TuJl+ cell body was found in the marginal zone of the Ixrαβ ^"7" mice, providing indication that while neurogenesis was partially impaired in the single mutants, it was severely impaired in the lxraβ ^'f~ mice.

Thus, combined, our results indicate that ablation of LXR function in the developing VM enhances gliogenesis and severely impairs neurogenesis by an overall decrease in dopaminergic neurogenic gene expression programs.

LXRs specifically mediate oxysterol effects on midbrain dopaminergic neurons

To further examine the function of LXRs, we combined gain and loss of function experiments and performed 22-hydroxycholesterol treatments in primary VM progenitor cultures from wild-type or LXRαβ-/- Ell embryos. Treatment of LXRcφ-/- VM progenitors with 22- hydroxycholesterol failed to enhance the number of dopaminergic neurons, while it increased the number of TH+ cells in wildtype cultures. (Figure 17A-D and Figure 18) . Thus, our data shows that oxysterols activate intracellular LXR receptors to specifically promote VM dopaminergic neurogenesis.

Effects of Liver-X-receptor (LXR) ligands on dopaminergic and neuronal differentiation of hES.

To investigate the effects of exogenously administered LXR ligands on dopaminergic differentiation of hES cells, we tested different concentrations of 22-R-hydroxycholesterol (oxysterol, natural ligand of LXR) . In the control group the percentage of TuJl+ neurons after differentiation was 18.90 ± 0.48% in HS181 cell line, and 20.0 ± 0.53% in H9 cell line. Treatment with 22-R-cholesterol (0.1 to 1 μM) significantly increased the percentage of TuJl generated neurons in both cell lines. This increase was superior to 50% in the best cases (p < 0.01, by ANOVA with post hoc Dunnett's comparisons).

However, this effect was bimodal and high concentrations of oxysterol (5μM) was toxic for the cells, resulting in abundant cell death at the end of the differentiation. The percentage of TH+ neurons in the control group was 4.13 ± 0.82% in HS181 cell line and 4.47 ± 0.38% for the H9 cell line, oxysterol treatment had an important effect on TH generation, increasing 3 to 4 times the number of TH+ neurons as compared to controls, at concentrations between 0.1 to 1 μM . This increase was similar in both cell lines. (Figures 20A-D and Figure 21A-D ) . All TH+ cells showed co- localization with TuJl antigen, confirming their neuronal nature. A notable effect on TH differentiation was also observed when we analyzed the percentage of TH+ neurons related to total of TuJl+ neurons. In controls around 20% of TuJl+ neurons were also TH+. 22- R-hydroxycholesterol treatment (0.1 to 1 μM) significantly increased this percentage, with values of approx. 60% of the total neurons coexpressing TH+ (Figures 2OD and 21D) .

Effects of Liver-X-receptor β (LXRβ) and LXR ligands on dopaminergic and neuronal differentiation of mES cells Having shown that LXR ligands are capable of regulating and promoting DA neurogenesis, we set to determine whether the presence of LXRβ was also limiting. We therefore set to examine whether overexpression of LXRβ per se, or in combination with LXR ligand treatment, could further enhance the dopaminergic differentiation of stem cells. We first examined the effects of the LXR ligand (22-R- hydroxycholesterol, 0.5 uM) on the differentiation of mES and confirmed that LXR ligands also increase the neuronal and dopaminergic differentiation of mouse ES cells.

We observed that the treatment with oxysterol of differentiating control cultures from day 8 of differentiation, significantly increased the percentage of TH+ colonies (p<0.05) and the number of TH+ cells per surface Unit inside the colonies, as compared with controls not treated (p < 0.01). We then performed similar

experiments with mES cells stably overexpressing LXRβ . For that we used vectors expressing: Empty-FLAG (as a control) , or LXRβ-FLAG. Interestingly, we found that mES cells overexpressing the LXRβ receptor showed increased dopaminergic differentiation as compared to controls, both at level of percentage of TH+ colonies and the number of TH+ cells/area (p< 0.01) (Figure 22A-D) . This last parameter, TH+ cells/area, was even higher when these cells were treated with 22-R-hydroxycholesterol during the differentiation period. The percentage of TuJl+ colonies was almost 100% in all the groups, and was not changed by the different treatments, although changes in the numbers of TuJl+ cells per colony were noted (Figure 22D) .

Effects of Oxysterols on Dopaminergic Neurogenesis and Differentiation of Human Embryonic Stem Cells

In order to verify the midbrain identity and the dopaminergic phenotype of the TH+ cells generated from the hES cells by differentiation on PA6, we examined the expression of different markers in H9 cells (passage 35) and HS181 (passage 40) . TH+ cells were found to express the transcription factors engrailedl and nurrl, the dopaminergic marker VMAT, and the GIRK2 potassium channel, typical of nigral neurons. Very few cells stained positive for with anti-GABA antibodies. Glial cells were present in the cultures and nestin+ cells were abundant, suggesting that several of the cells were still undifferentiated. Thus, our findings indicate that both cell lines respond in a very similar manner to differentiation on PA6 and give rise to high numbers of TH+ cells with reproducible phenotype .

We next compared the standard PA6 differentiation protocol with our protocol in which we introduced oxysterol treatment. As reported above, we found increased numbers of TH+ cells that were GABA negative, but expressed engrailed, providing indication that they are midbrain DA neurons. Oxysterol treatment enhanced the levels of

VMAT in the cytosol and engrailedl in the nuclei of TH+ cells. We also detected a high number of TH+ cells double-positive for DAT and GIRK2. Interestingly, we did not detect TH+/Calbindin+ cells, indicating that the phenotype generated in vitro was mainly of a nigral type. Finally, very few cells stained for DBH, GABA or 5-HT, indicating that the cultures contain very few noradrenergic, GABAergic or serotonergic neurons.

Having shown that LXR ligands are capable of regulating and promoting DA neurogenesis, we set to investigate whether the expression of LXRβ in the stem cells was also a limiting factor. We therefore examined whether overexpression of LXRβ per se, or in combination with LXR ligand treatment, could further enhance the dopaminergic differentiation of stem cells. We first examined the effects of the LXR ligand (22-R-hydroxycholesterol, 0.5 uM) on the differentiation of mES and confirmed that LXR ligands also increase the neuronal and dopaminergic differentiation of mouse ES cells. We observed that the treatment with oxysterol of differentiating control cultures from day 8 of differentiation, significantly increased the percentage of TH+ colonies (p<0.05) and the number of TH+ cells per surface Unit inside the colonies, as compared with controls not treated (p < 0.01). We then performed similar experiments with mES cells stably overexpressing LXRβ. For that we used vectors expressing: Empty-FLAG (as a control) , or LXRβ-FLAG. Interestingly we found that mES cells overexpressing the LXRβ receptor showed increased dopaminergic differentiation as compared to controls, both at the percentage of TH+ colonies and the number of TH+ cells/area (p< 0.01). Moreover, treatment of mES cells over- expressing LXRβ with 22-R-hydroxycholesterol during the differentiation period increased the number of TH+ cells/area to a greater extent than overexpression alone or oxysterol alone. The percentage of TuJl+ colonies was almost 100% in all the groups, and was not changed by the different treatments, although changes in the numbers of TuJl+ cells per colony were noted.

Discussion

Oxysterol ligands stimulate dopaminergic neurogenesis -To define the developmental processes affected by LXR ligands, we utilized both primary cortical and ventral mesencephalic precursor cultures. Treatment with exogenous oxysterol ligands enhanced TH+ dopaminergic differentiation exclusively in ventral midbrain precursor cultures. Thus, our results provide indication that despite the broad expression pattern of LXRs, the effects of oxysterols on midbrain dopaminergic differentiation were region- specific. In line with this, we found that the increase in TH+ DA neurons induced by oxysterols was completely abolished in LXRαβ-/- VM precursor cultures, indicating that intracellular LXRs are required to mediate the effects of oxysterols.

The finding that oxysterols regulate DA neurogenesis opens the door for the possible application of LXRs to promote DA neurogenesis in stem cell-based cultures for cell replacement strategies, for example for the treatment of neurogenerative disorders, such as Parkinson's disease.

Impaired neurogenesis and enhanced progenitor proliferation and gliogenesis in the VM of LXR knockout mice

LXR null mice displayed increased numbers of both BrdU+ and phospho- histone H3+ proliferating progenitors in the VM neuroepithelium. These observations were particularly evident in LXRαβ-/- mice compared to LXRα-/- and LXRβ-/- mice, providing indication that the function of LXRα and β are to some extent redundant. The increased numbers of proliferating progenitors in the neuroepithelium of LXR null mice were accompanied by a down-regulation in several prototypical markers for midbrain DA neurons, including Nurrl, Pitx3 and Wntl, and neurogenic genes such as Ngn2, and Hes5 and Dill, two regulators of Notch signals that modulate proneural activity. Additionally we observed decreased levels of Tis21, an anti-

proliferative gene expressed in neuroepithelial cells/radial glial preparing for neurogenic divisions.

We also analyzed LXR null mice for the expression of two recently identified DA cell fate determinants, the homeodomain proteins Lmxla and Msxl. However, no alterations in the levels of Lmxla or Msxl mRNAs were detected in any of the LXR genotypes. Surprisingly however, a dramatic loss in the number of TH+ DA neurons and Tujl+ neurons was detected in the ventral midbrains of LXR null mice. Moreover, these defects resulted in permanent losses in the number of TH+ neurons at birth. Given that no increase in cell death was detected and most proneural markers in the VMs of LXR null mice were downregulated, our results indicate that the DA deficit involves the loss of positive regulators of neurogenesis.

In addition to neurogenic deficits, we also observed that LXR null mice displayed a significant increase in the number of RC2+ radial glia at E13.5 and of GFAP+ cells at Pl. In fact, precocious GFAP transcripts were detected as early as Ell in LXRαβ-/- mice. Interestingly, at this stage, LXR null mice also expressed higher levels of Lmxlb and Raldhlal, two genes expressed in the VM by cells with radial glia morphology. Thus, our data provide indication that LXRs, unlike Ngn2, normally function to repress precocious gliogenesis in the VM. Combined, our results provide indication that despite a correct DA cell type specification in the LXR nulls, an insufficient number of progenitors are recruited .towards a neuronal fate and excess glial cells are prematurely generated. Thus, LXRs play a decisive role in specifying neuronal versus glial fate in VM progenitors .

Regulation of neuron vs glia fate decisions by LXRs in the VM Our in situ hybridization studies show an overlapping pattern expression of LXRα and LXRβ in the VM. Moreover, the phenotypes of LXRα-/- mice and LXRβ-/- mice were qualitatively similar {both

showed reduced neurogenesis and increased gliogenesis) . LXRβ-/- mice displayed a quantitatively more severe phenotype that LXRα-/- mice, providing indication that LXRβ is the preferred receptor in the developing VM. However, the phenotypes of LXRα-/- and LXRβ-/- mice and their effects on gene regulation were additive with the following order of severity; LXRαβ-/-> LXRβ-/-> LXRα-/->wildtype . Thus, our results support the hypothesis that the two receptors contribute to regulate glial versus neuronal fate decisions in the developing VM.

However, a complete ablation of TH+ neurons was not observed in any of the genotypes analyzed, providing indication that additional mechanisms regulate DA neurogenesis. In agreement with this observation, we recently found that DA neurogenesis was severely impaired in the Ngn2 nulls and that Mashl could partially compensate for the loss of Ngn2 11. Our results showing that Ngn2, but not Mashl mRNA levels, were reduced in LXRαβ-/- mice, raise the possibility that Mashl could also partially compensate for the loss of TH+ neurons in the VM of LXR nulls by 13.5 (Figure 6) .

In our study, we also describe that LXRs form heterodimers with RXR to regulate transcription in DA cells, regulate the expression of a number of genes in vivo that have not been previously described, and regulate neural versus giia fate decisions. Interestingly, LXRs are known to work as transcriptional repressors through the nuclear receptor corepressor (NCoR) and the silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) .

The phenotype of LXR null animals resembles that of the N-CoR-/- mice, where cortical progenitors prematurely differentiate into astroglia and neurogenesis is reduced. In line with a role of LXR as a repressor, we found an upregulation of GFAP and an increase in the number of radial glia and astrocytes in the LXR mutants, providing indication that LXRs primarily block gliogenesis. However, we also

found that deletion of LXRs resulted in the downregulation of several genes involved in neurogenesis and decreased numbers of DA neurons. These findings lead us to hypothesize that LXRs may indirectly activate neurogenesis by repressing the negative regulator/s of proneural genes (Dill, Hes5, Ngn2) , and of genes regulating neurogenic division (Tis21) or DA neurogenesis (Wntl) . Thus, in the absence of LXRs, neurogenesis ceases and gliogenesis proceeds prematurely, despite the normal expression of DA cell fate determinants (Lmxla and Msxl) .

In summary, the generation of cell-type diversity in the developing CNS is a highly regulated process, coordinated by the expression of specific morphogens and transcription factors that result in the sequential formation of sufficient neurons and glia. This application provides the first genetic evidence that two nuclear receptors, the liver X receptors α and β, exert a novel function in the brain: regulating a binary cell-fate choice between neuron and glia in progenitor cells. Our data support a model in which LXRs maintain radial glia while promoting neurogenesis and repressing gliogenesis in vivo. These observations provide indication that LXRs control the proportion of progenitors recruited to neurogenic or gliogenic programs by regulating gliogenic and neurogenic networks.

Our results show that oxysterols alone or combined with overexpression of LXR receptors enhance the generation of dopaminergic neurons from mouse or human embryonic stem cells . Administration of oxysterols decreases progenitor proliferation and gliogenesis and enhances neurogenic divisions, resulting in increases numbers of neurons and decreased numbers of glial cells. This decreases the number of gial cells and produces a pan-neuronal phenotype, with a particularly severe alteration of the DA system. Deletion of LXRαβ increases proliferative divisions and gliogenesis in neuronal progenitors and reduces neurogenic divisions.

Endogenous levels of LXR receptors and ligands, despite being expressed and required for normal ventral midbrain development in vivo, are limiting and not sufficient for optimal differentiation of stem cells in vitro. This finding opens the door to different avenues of therapeutic intervention, for diseases such as

Parkinson's disease, Huntington' s .disease, brain cancer and stroke.

Our results show that LXR receptor/ligands may be used to enhance neurogenesis and decrease proliferation and may be applied in vitro, in stem/progenitor cell preparations, and also in vivo, in circumstances in which it is necessary to induce neurogenesis/neuronal differentiation of endogenous or grafted stem/progenitor cells. Furthermore, LXR receptor/ligands are shown herein to enhance the generation of neurons other than dopaminergic neurons, such as motor neurons. The approaches described herein may therefore be useful in the treatment of diseases such as stroke and motor neuron disease.

In vivo, LXR ligands can be either delivered orally, since they cross the blood-brain barrier and reach the central nervous system, or via an infusion cannula. Administration of LXR ligands may be a valuable tool to enhance neurogenesis and dopaminergic differentiation in endogenous or exogenously supplied stem/progenitor cells. Moreover, the cellular mechanism and the signalling pathways activated by LXR differ from other glia-derived pro-dopaminergic factors that we have recently identified, such as Wnts. For example, the effects of LXR and Wnts could be either additive or complementary, which would allow to further enhance the survival and functional engraftment of dopaminergic neurons in animal models of neurodegenerative diseases such as Parkinson's disease in vivo.

LXR receptors and ligands may also be useful for promoting differentiation of stem cells into neurons. For example, LXR

receptors and ligands may be used in methods protocols involving stem cells, such as embryonic stem cells, in cell replacement therapies for neurodegenerative processes, such as Parkinson's disease or Huntington's disease. LXR receptors and ligands may also be useful for preventing the formation of tumours in neural tissue. LXR receptors and ligands may be used in the treatment of conditions associated with cell-loss and neurogenesis, such as stroke. LXR receptors and ligands may also be useful for increasing endogenous neurogenesis. For example, increased neurogenesis from the subventricular zone may the useful in the treatment of stroke and increased neurogenesis, of progenitors lining the ependymal canal may be useful in the treatment of motor neuron diseases .

References

Arenas E. (2005) Ann N Y Acad Sci. 1049:51-56.

Andersson E, et al (2006) Cell 124: 393-405.

Andersson, E et al Development 133, 507-16 (2006) . Andersson S et al (2005) Proc Natl Acad Sci USA. 102: 3857-3862.

Barberi T et al (2003) Nat Biotechnol. 21(10): 1200-7.

Edwards PA et al (2002) J Lipid Res . 43: 2-12.

Kawasaki H et al (2000) Neuron 28(1): 31-40.

Kim JH et al (2002) Nature. 418:50-6. KeIe, J. et al . Development 133, 495-505 (2006).

Lee SH et al (2000) Nat Biotechnol. 18: 675-9.

Lee SM, et al Development. 1997 Mar; 124 (5) : 959-69.

Lindvall 0 et al (2004) Nat Med. 10 Suppl: S42-50.

Lu TT et al (2001) J Biol Chem. 276: 37735-38. Maxwell SH and Li M. (2005) J Anat. 207: 209-218.

Perrier AL et al . (2004) PNAS USA. 101:12543-8.

Roy N et al (2006) Nat. Med. Doi: 10.1038/nml495.

Smidt, M. P. et al . Nat Neurosci 3, 337-41 (2000).

Smidt, M. P. et al . Development 131, 1145-55 (2004) . Snyder B. et al (2005) Current Opinion in Neurobiology 18: 376-385.

Tamehiro N et al (2005) FEBS Lett. 579: 5299-5304

Taylor H et al (2005) Current Opinion in Biotechnology 16: 1-6.

Wang L et al (2002) PNAS USA. 99: 13878-83.

Yan Y et al (2005) Stem Cells. 23: 781-90. Ye, W. et al. Cell 93, 755-66 (1998).

Thomas, K. R et al Nature 346, 847-50 (1990) .

McMahon, A. P. et al Cell 62, 1073-85 (1990) .

Castelo-Branco, G. et al . PNAS U S A 100, 12747-52 (2003).

Prakash, N. et al . Development 133, 89-98 (2006) . Vernay, B. et al . J Neurosci 25, 4856-67 (2005).

Puelles, E. et al . Development 131, 2037-48 (2004).

Castillo, S. O. et al . MoI Cell Neurosci 11, 36-46 (1998).

Saucedo-Cardenas, O. et al . PNAS U S A 95, 4013-8 (1998).

Zetterstrom, R. H. et al. Science 276, 248-50 (1997).

Wagner, J. et al. Nat Biotechnol 17, 653-9 (1999).

Kim, J. H. et al . Nature 418, 50-6 (2002).

Kim, J. Y. et al .. J Neurochem 85, 1443-54 (2003).

Wallen-Mackenzie, A. et al Genes Dev 17, 3036-47 (2003) . Diez del Corral, R. et al Neuron 40, 65-79 (2003) .

Krezel, W. et al . Science 279, 863-7 (1998).

Wong, L. F. et al .. Nat Neurosci 9, 243-50 (2006).

Schultz, J. R. et al. Genes Dev 14, 2831-8 (2000).

Venkateswaran, A. et al PNAS U S A 97, 12097-102 (2000) . Alberti, S. et al . J Clin Invest 107, 565-73 (2001).

Joseph, S. B et al. Nat Med 9, 213-9 (2003).

Janowski, B. A. et al Nature 383, 728-31 (1996) .

Willy, P. J. et al Genes Dev 11, 289-98 (1997) .

Willy, P. J. et al.. Genes Dev 9, 1033-45 (1995). Repa, J. J. et al. J Biol Chem 277, 18793-800 (2002).

Zelcer, N. et al. J Clin Invest 116, 607-14 (2006) .

Andersson, S., et al Proc Natl Acad Sci U S A 102, 3857-62 (2005).

Wang, L. et al . PNAS U S A 99, 13878-83 (2002). _.

Mo, J., et al J Biol Chem 277, 50788-94 (2002). Sacchetti, P., et al J Biol Chem 277, 35088-96 (2002).

Haubensak, W et al PNAS U S A 101, 3196-201 (2004) .

Kosodo, Y. et al. Embo J 23, 2314-24 (2004).

Corcoran, R. B et al PNAS U S A 103, 8408-13 (2006) .

Komuves, L. G. et al J Invest Dermatol 118, 25-34 (2002) . Castelo-Branco, G. et al. MoI Cell Neurosci 31, 251-62 (2006).

Bjorkhem, I. et al Arterioscler Thromb Vase Biol 24, 806-15 (2004)

Malatesta, P et al Development 127, 5253-63 (2000) .

Malatesta, P. et al Neuron 37, 751-64 (2003) .

Hu, X., et al MoI Endocrinol 17, 1019-26 (2003). Wagner, B. L. et al . MoI Cell Biol 23, 5780-9 (2003).

Jepsen, K. et al . Cell 102, 753-63 (2000).

Hermanson, 0., et al. Nature 419, 934-9 (2002).

Danielian PS, McMahon AP. Nature. (1996) ; 383 (6598) : 332-4.

Thomas KR, Capecchi MR. Nature. 1990 Aug 30/346 (6287) : 847-50.

Matsunaga E, et al Development. 2002 Nov; 129 (22) :5269-77.