WO2008071960A2 - Procédé permettant d'augmenter la neurogenèse - Google Patents

Procédé permettant d'augmenter la neurogenèse Download PDF

Info

Publication number
WO2008071960A2
WO2008071960A2 PCT/GB2007/004765 GB2007004765W WO2008071960A2 WO 2008071960 A2 WO2008071960 A2 WO 2008071960A2 GB 2007004765 W GB2007004765 W GB 2007004765W WO 2008071960 A2 WO2008071960 A2 WO 2008071960A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
lxr
stem
cells
neural
Prior art date
Application number
PCT/GB2007/004765
Other languages
English (en)
Other versions
WO2008071960A3 (fr
Inventor
Ernest Arenas
Anita Hall
Paola Sacchetti
Kyle Sousa
Isabel Liste
Original Assignee
Neuro Therapeutics Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neuro Therapeutics Ab filed Critical Neuro Therapeutics Ab
Publication of WO2008071960A2 publication Critical patent/WO2008071960A2/fr
Publication of WO2008071960A3 publication Critical patent/WO2008071960A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton

Definitions

  • This invention relates to the promotion of neurogenesis, in particular dopaminergic neurogenesis in progenitor or precursor cells.
  • Parkinson's disease 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.
  • 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.
  • stem cells have the ability to differentiate into neural cell lineages including neurons, astrocytes and oligodendrocytes.
  • 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 .
  • RG radial glial
  • 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.
  • MHO midbrain-hindbrain organizer
  • 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).
  • Otx2 the homeodomain transcription factor
  • FGF8 Prakash et al., 2006
  • Lmxlb the homeodomain transcription factor
  • 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).
  • LXRs liver X receptors
  • RXR retinoid X receptor
  • LXR ⁇ is ubiquitously expressed, the expression of LXR ⁇ is limited to the liver, kidney, intestine, adipose tissue and macrophages (Lu et al . , 2001).
  • Mice with inactivated LXR ⁇ 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).
  • LXR ligands and receptors promote neurogenesis.
  • 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,
  • 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
  • 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.
  • 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.
  • suitable binding partners include LXR ligands.
  • a LXR receptor may be an LXR ⁇ or a LXR ⁇ receptor from any mammalian species, for example a mouse LXR ⁇ or LXR ⁇ receptor or human LXR ⁇ or LXR ⁇ 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.
  • 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.
  • 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 LXR ⁇ or LXR ⁇ 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 LXR ⁇ or LXR ⁇ 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 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.
  • 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).
  • 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)
  • 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
  • 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,
  • 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 .
  • RXR retinoid X receptor
  • 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.
  • 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
  • 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 ubiquitin
  • 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.
  • a cell may be treated with two or more LXR activators, for example a LXR ligand and a LXR receptor.
  • LXR activators for example a LXR ligand and a LXR receptor.
  • 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.
  • 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.
  • 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.
  • a LXR suppressor may selectively down-regulate the expression of LXR ⁇ or LXR ⁇ . Down regulation may occur, for example, through RNA interference (RNAi) .
  • RNAi RNA interference
  • 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.
  • siRNAs small interfering RNAs
  • PTGs post transcriptional gene silencing
  • miRNAs micro-RNAs
  • targeted transcriptional gene silencing targeted transcriptional gene silencing.
  • Double-stranded RNA (dsRNA) -dependent post transcriptional silencing also known as RNA interference (RNAi)
  • RNAi RNA interference
  • 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.
  • 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 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.
  • the present invention provides the use of these sequences as LXR suppressors for downregulating the expression of
  • LXR ⁇ or LXR ⁇ are 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.
  • 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.
  • the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo) nucleotides, typically a UU of dTdT 3' overhang.
  • siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors) .
  • expression systems e.g. vectors
  • 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.
  • 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).
  • 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.
  • the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression.
  • 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.
  • the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell.
  • the shRNA molecule comprises a partial sequence of LXR ⁇ and LXR ⁇ mRNA.
  • 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.
  • the siRNA molecule, longer dsRNA molecule or miRNA molecule comprises a partial sequence of the LXR ⁇ or LXR ⁇ mRNA.
  • 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.
  • expression of the RNA sequence can be regulated using a tissue specific promoter.
  • the siRNA, longer dsRNA or miRNA is produced exogenously (in vitro) by transcription from a vector.
  • the vector may comprise a nucleic acid sequence in both the sense and antisense orientation, such that, when
  • the sense and antisense sections will associate to form a double stranded RNA.
  • the vector comprises LXR ⁇ or LXR ⁇ nucleic acid sequences; or variants or fragments thereof.
  • the sense and antisense sequences are provided on different vectors.
  • 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
  • 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- .
  • RNAi molecules for down-regulation of LXR ⁇ or LXR ⁇ 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 LXR ⁇ or LXR ⁇ 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.
  • a compound such as an LXR activator or suppressor
  • 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.
  • cytochrome P-450 enzymes 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.
  • 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.
  • 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.
  • 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.
  • 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 badly 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).
  • a cell which is Nurrl negative may spontaneously express Nurrl during the differentiation process.
  • 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.
  • a majority of the cells may be induced to adopt a neuronal fate.
  • 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.
  • dopaminergic induction or differentiation may be enhanced in neuronal cells.
  • a majority of the cells may be induced to adopt a glial fate.
  • 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.
  • 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.
  • 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.
  • 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.
  • an inducible promoter is the Tetracyclin ON/OFF system (Gossen, et al . , 1995) in which gene expression is regulated by tetracyclin analogs.
  • 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.
  • 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 .
  • 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
  • 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 .
  • expression of LXR activator above basal levels may be caused by introduction of one or more extra copies of the LXR activator into
  • 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.
  • 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.
  • 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 .
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • RXR retinoid X receptor
  • 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.
  • a member of the FGF family of growth factors e.g. FGF4, FGF8 or FGF20
  • the cells may be contacted with two or more of the above agents .
  • 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.
  • 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.
  • 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) .
  • 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.
  • LRP L_ow density lipoprotein-receptor irelated p_roteins
  • 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).
  • IP3 inositol 1,4,5 triphosphate
  • 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.
  • 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.
  • 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.
  • 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, ⁇ -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.
  • expression of tyrosine hydroxylase (TH) , aromatic L-amino acid decarboxylase (AADC) , dopamine transporter (DAT) and dopamine receptors may be used.
  • 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)
  • 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.
  • GFAP glial fibrilary acidic protein
  • vimentin a protein that influences the expression of the cell
  • RC2 a protein that influences the expression of the cell
  • NG2B5 glial fibrilary acidic protein
  • 04 a protein that influences the expression of the cell
  • RIP a protein that influences the expression of the cell
  • BLBP Brain lipid binding protein
  • MBP myelin basic protein
  • 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.
  • 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.
  • 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% S
  • 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.
  • the neuron may have a particular- neuronal phenotype.
  • the neuron has a dopaminergic phenotype.
  • 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.
  • 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.
  • 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.
  • the factor or expression of the encoded molecule may function in neuroprotection or neuroregeneration of the cellular environment surrounding that neuron.
  • 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.
  • 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
  • GDNF glial-derived neurotropic growth factor
  • GDNF 5 hydroxylase expression
  • Tomac et al. (1995) Nature, 373, 335-339; Arenas, et al . , (1995) Neuron, 15,1465-1473.
  • Other neurotropic molecules of the GDNF family include Neurturin, Persephin and Artemin .
  • Neurotropic molecules of the neurotropin family include nerve growth factor (NGF) , brain derived neurotropic factor (BDNF) , and neurotropin-3, -4/5 and -6.
  • Other factors with neurotrophic activity include members of the FGF family for instance FGF2, 4, 8 and 20; members of the Wnt family, including Wnt-1, -2, -5a, -3a and 7a;
  • BMP2 5 members of the BMP family, including BMP2, 4, 5 and 7, nodal, activins and GDF; and members of the TGFalpha/beta family.
  • 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.
  • 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.
  • 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).
  • Ngn2 Kelle et al . , 2006, Anderson et al., 2006, Development
  • 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) .
  • FACS fluorescence activated cell sorting
  • 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, RPTP ⁇ /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.
  • 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.
  • 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.
  • 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 .
  • Methods of the invention may comprise additional negative or positive selection methods to enrich for neural stem, progenitor or precursor cells, or other stem or neural cells with the desired phenotype .
  • Negative selection may be used to enrich for target cells, for example, glial cells or neurons, such as dopaminergic neurons.
  • target cells for example, glial cells or neurons, such as dopaminergic neurons.
  • neurotoxins for non-DA neurons for instance 5-7-dihydroxytryptamine
  • 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
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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) .
  • an antioxidant e.g. ascorbic acid
  • low oxygen tension e.g. ascorbic acid
  • 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
  • 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.
  • 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.
  • 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.
  • SCNT somatic cell nuclear transfer
  • 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.
  • 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.
  • 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 .
  • one or more methods of the invention may be performed to prepare cells for administering into an individual.
  • 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.
  • Wnt-5a 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.
  • an LXR activator such as an LXR ligand or LXR receptor as described herein
  • 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.
  • 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) .
  • NTN neurturin
  • BDNF brain-derived neurotrophic factor
  • 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
  • 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.
  • 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.
  • a neuroprotective molecule 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.
  • the method of making a pharmaceutical composition may include admixing the cell with one or more factors found in the developing ventral mesencephalon.
  • 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.
  • 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.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection.
  • a composition may be prepared using artificial cerebrospinal fluid.
  • 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.
  • 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).
  • 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 .
  • a “prophylactically effective amount” or a “therapeutically effective amount” as the case may be, although prophylaxis may be considered therapy
  • 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.
  • 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.
  • 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.
  • one or more MHC alleles in a donor cell may be replaced with those of a recipient, e.g. by homologous recombination.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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
  • 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
  • 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 polymerase chain reaction
  • 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.
  • 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.
  • 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.
  • a detectable signal preferably a visually detectable signal, such as a coloured product.
  • Many examples are known, including ⁇ -galactosidase and luciferase.
  • ⁇ -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.
  • a method of identifying a compound useful in promoting neurogenesis or gliogenesis may comprise:
  • 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.
  • 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.
  • an LXR activator such as an LXR ligand, for example an oxysterol
  • 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.
  • 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.
  • a LXR suppressor for example an anti- LXR RNAi molecule
  • 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.
  • 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 neurite
  • 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:
  • 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:
  • test compounds 0 or more test compounds
  • 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 .
  • 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.
  • LXR activator is a compound which increases the amount of LXR signalling in a cell. LXR activators are described in more detail above .
  • 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
  • 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:
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ M, e.g. 0.1 to 50 ⁇ M, such as about 10 ⁇ M. 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.
  • 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.
  • Cholesterol derivatives may be employed as mimetics. Cholesterol derivatives may be produced, for example, by hydroxylation or the addition of acid groups.
  • 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.
  • 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
  • composition 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.
  • 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.
  • 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.
  • 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, LXR ⁇ " and LXR ⁇ " /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 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”.
  • 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 .
  • a nucleic acid array may comprise nucleotide sequences.
  • 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.
  • 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.
  • 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
  • Suitable labels include fluorescent labels, such as cyanine 3 or cyanine 5.
  • the labelled extension products
  • 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.
  • Figure 1 shows that Liver X receptors are expressed during dopaminergic neurogenesis.
  • A-B Quantitative PCR analysis revealed that LXR ⁇ and LXR ⁇ 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 LXR ⁇ , LXR ⁇ , and RXR ⁇ 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.
  • FIG. 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, LXR ⁇ and RXR ⁇ (B) and LXR ⁇ and RXR ⁇ (C) , as indicated.
  • LXRE-Luc LXR binding sites
  • 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 lO ⁇ M 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 Brd ⁇ , a proliferation marker also decreased in ventral midbrain primary cells treated with LXR ligand (E-H) .
  • Treatment with lO ⁇ M 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 lO ⁇ M 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.
  • 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 LXR ⁇ and LXR ⁇ 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 LXR ⁇ null mice, while LXR ⁇ and LXR ⁇ null mice displayed losses of almost
  • 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 LXR ⁇ -/- and LXR ⁇ -/- 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).
  • 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.
  • FIG. 15 shows coronal sections through the midbrains of Pl wildtype and LXRc ⁇ -/- mice.
  • Pl LXR ⁇ -/- mice retain their dopaminergic deficits (TH) (A, B) and exhibit increased numbers of GFAP+ astrocytes throughout the ventral midbrain (C-F) .
  • TH dopaminergic deficits
  • A, B dopaminergic deficits
  • C-F ventral midbrain
  • 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.
  • Tis21 a marker of neurogenic asymmetric division was also significantly reduced in the VM of LXR ⁇ nulls (K) .
  • Precocious expression of the astrocytic marker GFAP was observed, particularly in LXR ⁇ and LXR ⁇ 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 LXRot ⁇ -/- 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
  • Figure 20 shows oxysterols increase dopaminergic neuron
  • hES cells line H9 on passage 2 of dopaminergic differentiation were treated with different concentrations of 22-R-hydroxycholesterol (Control, 0,l ⁇ M, 0,5 ⁇ M, l ⁇ M and 5 ⁇ M) .
  • D Percentage of TH-positive cells among all TuJl- positive neurons. (*) p ⁇ 0.05; (**) p ⁇ 0.01.
  • Figure 21 shows oxysterols increase dopaminergic neuron
  • hES cells line HS181 on passage 2 of dopaminergic differentiation were treated with different concentrations of 22-R-hydroxycholesterol (Control, 0,l ⁇ M, i ⁇ M and 5 ⁇ M) .
  • 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.
  • Differentiating mES (on day 8) were treated with/without the oxysterol 22-R-hydroxycholesterol at 0,5 ⁇ M until de end of the differentiation.
  • B, C) Represent the percentages of TH-positive (B) and TuJl- positive colonies (C) among all colonies.
  • hES cell lines H9 WA-09, XX, passages 35-40
  • HS181 XX, passages 35-45
  • hFF mitotically inactivated human foreskin fibroblasts
  • hFF mitotically inactivated human foreskin fibroblasts
  • SRM Knockout serum replacement
  • bFGF basic fibroblast growth factor
  • 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 ⁇ /ml; Invitrogen) .
  • SRM Knockout serum replacement
  • 2mM L-glutamine Invitrogen
  • 1% nonessential amino acids Invitrogen
  • O.lmM beta-mercaptoethanol Sigma
  • LIF 1000 U/ml ESGRO, Chemicon
  • Pen./Strept. (10.000 ⁇ /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 ⁇ g/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 ⁇ iM L-glutamine. hFF and PA6 cells were mitotically inactivated before use, by treatment with mitomycin C (l ⁇ g/ml; Roche) for 2-4 h at 37°C. All cell lines were maintained at 37°C, 5% CO 2 and 95% humidity.
  • Invitrogen alpha-minimum essential medium
  • hFF and PA6 cells were mitotically inactivated before use, by treatment with mitomycin C (l ⁇ g/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. O ⁇ g of plasmid DNA/well complexed with 2 ⁇ l 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.
  • pRL-TK Renilla luciferase
  • lipid/DNA mix was replaced with fresh 2.5% serum medium containing vehicle or appropriate ligand (lO ⁇ M), 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.
  • hES 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 .
  • SRM serum replacement medium
  • FGF8 25-100 ng/ml; R&D Systems
  • 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.
  • mES 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.
  • SRM serum replacement medium
  • SRM serum replacement medium
  • FGF8 25-100 ng/ml; R&D Systems
  • 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 ⁇ m2) . Data from different groups were compared with ANOVA and Dunnett's Multiple Comparison post hoc analysis .
  • 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 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 ⁇ M sections of the entire ventral midbrain were cut on a cryostat.
  • LXRs are spatially and temporally expressed in proliferating progenitors of the developing ventral midbrain
  • 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.
  • 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.
  • bHLH basic-helix-loop-helix
  • Ngn2 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 LXRc ⁇ -/- mice, and severely reduced in LXR ⁇ -/- and LXR ⁇ -/- 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.
  • TuJ+ neurites crossing the midline followed a straight line, reflecting the presence of fewer cell bodies.
  • TuJl+ cell body was found in the marginal zone of the Ixr ⁇ "7" mice, providing indication that while neurogenesis was partially impaired in the single mutants, it was severely impaired in the lxra ⁇ 'f ⁇ mice.
  • LXR Liver-X-receptor
  • LXR ⁇ Liver-X-receptor ⁇
  • LXR ligands are capable of regulating and promoting DA neurogenesis
  • 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 .
  • LXR ligands are capable of regulating and promoting DA neurogenesis. We set to investigate whether the expression of LXR ⁇ in the stem cells was also a limiting factor. We therefore examined whether overexpression of LXR ⁇ 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.
  • LXR ligand 22-R-hydroxycholesterol, 0.5 uM
  • 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.
  • 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 LXR ⁇ -/- mice compared to LXR ⁇ -/- and LXR ⁇ -/- mice, providing indication that the function of LXR ⁇ and ⁇ 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.
  • LXR null mice displayed a significant increase in the number of RC2+ radial glia at E13.5 and of GFAP+ cells at Pl.
  • precocious GFAP transcripts were detected as early as Ell in LXR ⁇ -/- mice.
  • LXR null mice also expressed higher levels of Lmxlb and Raldhlal, two genes expressed in the VM by cells with radial glia morphology.
  • 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.
  • 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) .
  • NCoR nuclear receptor corepressor
  • SMRT retinoic acid and thyroid hormone receptors
  • LXR null animals resembles that of the N-CoR-/- mice, where cortical progenitors prematurely differentiate into astroglia and neurogenesis is reduced.
  • LXR a repressor
  • LXRs primarily block gliogenesis.
  • deletion of LXRs resulted in the downregulation of several genes involved in neurogenesis and decreased numbers of DA neurons.
  • 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) .
  • proneural genes Dill, Hes5, Ngn2
  • Tr21 neurogenic division
  • Wntl DA neurogenesis
  • 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 ⁇ and ⁇ , 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.
  • 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 LXR ⁇ increases proliferative divisions and gliogenesis in neuronal progenitors and reduces neurogenic divisions.
  • Parkinson's disease Huntington' s .disease, brain cancer and stroke.
  • 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.
  • 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.
  • 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.
  • Wnts glia-derived pro-dopaminergic factors that we have recently identified, such as Wnts.
  • 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.
  • 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 .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Psychology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne la stimulation de la neurogenèse dans des cellules progénitrices ou précurseurs grâce à la modulation de la quantité de signal du récepteur X hépatique (RXH) dans lesdites cellules. Selon l'invention, un signal RXH accru favorise la neurogenèse et un signal RXH réduit favorise la gliogenèse. Les procédés de l'invention peuvent se révéler utiles, par exemple, pour des thérapies de remplacement cellulaire destinées à traiter des pathologies caractérisées par une perte, une lésion ou un dysfonctionnement neuronal, telles que la maladie de Parkinson, la maladie d'Huntington, un accident vasculaire cérébral et une maladie des motoneurones, et ils peuvent également se révéler utiles pour l'étude des événements de signalisation dans les neurones et des effets de médicaments sur les neurones in vitro.
PCT/GB2007/004765 2006-12-12 2007-12-12 Procédé permettant d'augmenter la neurogenèse WO2008071960A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US87424506P 2006-12-12 2006-12-12
US60/874,245 2006-12-12

Publications (2)

Publication Number Publication Date
WO2008071960A2 true WO2008071960A2 (fr) 2008-06-19
WO2008071960A3 WO2008071960A3 (fr) 2009-05-28

Family

ID=39149413

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/004765 WO2008071960A2 (fr) 2006-12-12 2007-12-12 Procédé permettant d'augmenter la neurogenèse

Country Status (1)

Country Link
WO (1) WO2008071960A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012013936A1 (fr) 2010-07-30 2012-02-02 Cambridge Enterprise Limited Corticogénèse de cellules pluripotentes humaines
EP2457923A1 (fr) * 2009-07-24 2012-05-30 Kyowa Hakko Kirin Co., Ltd. Dérivé de stérol
US20160000807A1 (en) * 2013-02-27 2016-01-07 Ernest Arenas Compound and method for the treatment and diagnosis of neurodegenerative conditions
WO2016007762A1 (fr) * 2014-07-09 2016-01-14 Duke University Compositions et méthodes pour la réparation de la myéline
US11458146B2 (en) 2017-01-13 2022-10-04 Duke University Compositions and methods for the treatment of myelin related and inflammation related diseases or disorders

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003049685A2 (fr) * 2001-12-07 2003-06-19 The Regents Of The University Of California Traitement de la degenerescence maculaire liee au vieillissement
US20050003998A1 (en) * 2002-08-15 2005-01-06 Goran Bertilsson Therapeutic use of selective LXR modulators

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003049685A2 (fr) * 2001-12-07 2003-06-19 The Regents Of The University Of California Traitement de la degenerescence maculaire liee au vieillissement
US20050003998A1 (en) * 2002-08-15 2005-01-06 Goran Bertilsson Therapeutic use of selective LXR modulators

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANDERSSON SANDRA ET AL: "Inactivation of liver X receptor beta leads to adult-onset motor neuron degeneration in male mice." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 8 MAR 2005, vol. 102, no. 10, 8 March 2005 (2005-03-08), pages 3857-3862, XP007907041 ISSN: 0027-8424 *
FU X ET AL: "27-Hydroxycholesterol Is an Endogenous Ligand for Liver X Receptor in Cholesterol-loaded Cells" JOURNAL OF BIOLOGICAL CHEMISTRY 20011019 US, vol. 276, no. 42, 19 October 2001 (2001-10-19), pages 38378-38387, XP002513357 ISSN: 0021-9258 *
LEE CHANG SEOK ET AL: "Oxysterols suppress inducible nitric oxide synthase expression in lipopolysaccharide-stimulated astrocytes through liver X receptor" NEUROREPORT, vol. 17, no. 2, February 2006 (2006-02), pages 183-187, XP009111661 ISSN: 0959-4965 *
SZANTO ATTILA ET AL: "Transcriptional regulation of human CYP27 integrates retinoid, peroxisome proliferator-activated receptor, and liver X receptor signaling in macrophages." MOLECULAR AND CELLULAR BIOLOGY SEP 2004, vol. 24, no. 18, September 2004 (2004-09), pages 8154-8166, XP007907045 ISSN: 0270-7306 *
YOSHIDA T ET AL: "Cholestan-3beta,5alpha,6beta-triol, but not 7-ketocholesterol, suppresses taurocholate-induced mucin secretion by cultured dog gallbladder epithelial cells" FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 478, no. 1-2, 28 July 2000 (2000-07-28), pages 113-118, XP004337417 ISSN: 0014-5793 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2457923A1 (fr) * 2009-07-24 2012-05-30 Kyowa Hakko Kirin Co., Ltd. Dérivé de stérol
EP2457923A4 (fr) * 2009-07-24 2013-04-17 Kyowa Hakko Kirin Co Ltd Dérivé de stérol
JP5640009B2 (ja) * 2009-07-24 2014-12-10 協和発酵キリン株式会社 ステロール誘導体
US8952003B2 (en) 2009-07-24 2015-02-10 Kyowa Hakko Kirin Co., Ltd Sterol derivative
WO2012013936A1 (fr) 2010-07-30 2012-02-02 Cambridge Enterprise Limited Corticogénèse de cellules pluripotentes humaines
US20160000807A1 (en) * 2013-02-27 2016-01-07 Ernest Arenas Compound and method for the treatment and diagnosis of neurodegenerative conditions
US10226475B2 (en) * 2013-02-27 2019-03-12 Swansea University Compound and method for the treatment and diagnosis of neurodegenerative conditions
WO2016007762A1 (fr) * 2014-07-09 2016-01-14 Duke University Compositions et méthodes pour la réparation de la myéline
US10238664B2 (en) 2014-07-09 2019-03-26 Duke University Compositions and methods for the repair of myelin
US11426419B2 (en) 2014-07-09 2022-08-30 Duke University Compositions and methods for the repair of myelin
US12029742B2 (en) 2014-07-09 2024-07-09 Duke University Compositions and methods for the repair of myelin
US11458146B2 (en) 2017-01-13 2022-10-04 Duke University Compositions and methods for the treatment of myelin related and inflammation related diseases or disorders

Also Published As

Publication number Publication date
WO2008071960A3 (fr) 2009-05-28

Similar Documents

Publication Publication Date Title
US20230323294A1 (en) Differentiation of cortical neurons from human pluripotent stem cells
US20090087417A1 (en) Methods for promoting dopaminergic neuronal development by using ng4a-subfamily and wnt-ligands
US8470594B2 (en) Methods for identifying agents that affect the survival of motor neurons
Benkler et al. Altered astrocytic response to activation in SOD1G93A mice and its implications on amyotrophic lateral sclerosis pathogenesis
JP2006517084A (ja) 胚性幹細胞の分化を誘導する方法及びその用途
Grudina et al. Human NPCs can degrade α–syn fibrils and transfer them preferentially in a cell contact-dependent manner possibly through TNT-like structures
Kurowska et al. Intracellular Nogo-A facilitates initiation of neurite formation in mouse midbrain neurons in vitro
WO2021224496A1 (fr) Procédés de différenciation de cellules souches en cellules progénitrices dopaminergiques
JP2002542818A (ja) ニューロンの発生に関連する材料及び方法
WO2008071960A2 (fr) Procédé permettant d'augmenter la neurogenèse
EP1827470A2 (fr) Materiels et methodes lies aux ligands dickkopf et a la neurogenese
US20090226536A1 (en) Methods and materials relating to enhanced production of dopamine neurons
Corvaglia et al. ProNGF is a cell-type-specific mitogen for adult hippocampal and for induced neural stem cells
Bianco et al. Rapid serum-free isolation of oligodendrocyte progenitor cells from adult rat spinal cord
Yeon et al. Differentiation of astrocytes with characteristics of ventral midbrain from human embryonic stem cells
Kumar Characterization of Mouse and Human Astrocytes in Amyotrophic Lateral Sclerosis: Effects of Oxidative Stress and Blockade of the Metabotropic Glutamate Receptor 5
Fellows Investigating the role of the insulin-like growth factor receptor (IGF1R) on axonal transport as a target for pharmacological intervention in Amyotrophic Lateral Sclerosis
Ishan et al. Taste papilla cell differentiation requires tongue mesenchyme via ALK3-BMP signaling to regulate the production of secretory proteins
Ostick Investigating the effects of tau mutations in induced pluripotent stem cell-derived neurons
JP2009532057A (ja) 神経細胞の生存および発生
AU2004202661B2 (en) Materials and methods relating to neuronal development
Mishra Modeling neurodevelopment and cortical dysfunction in SPG11-linked hereditary spastic paraplegia using human induced pluripotent stem cells
Baptista Developing assays to assess structure and function of synapses in vitro
Glière Cinq esquisses, op. 17
Zuber et al. Recent progress in histochemistry

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07848509

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07848509

Country of ref document: EP

Kind code of ref document: A2