Title: Screening and
therapeutic methods relating to neurogenesis
United States Patent: 7,323,334
Issued: January 29, 2008
Inventors: Zhou; Qun-Yong
(Irvine, CA), Cheng; Michelle Y. (Irvine, CA)
Assignee: The Regents of
the University of California (Oakland, CA)
Appl. No.: 10/680,554
Filed: October 3, 2003
Master of Science in Law
The invention provides methods of
identifying compounds that modulate neurogenesis. The methods involve
providing a compound that modulates prokineticin receptor signaling;
contacting a neural stem or progenitor cell with the compound; and
determining the ability of the compound to modulate neurogenesis. The
invention also provides methods for modulating neurogenesis. The methods
involve contacting a neural stem or progenitor cell with an effective
amount of a compound that modulates prokineticin receptor signaling. Such
methods are useful for both ex vivo or in vivo therapeutic applications
where neural regeneration is desirable.
Description of the
SUMMARY OF THE INVENTION
The invention provides methods of identifying compounds that modulate
neurogenesis. The methods involve providing a compound that modulates
prokineticin receptor signaling; contacting a neural stem or progenitor cell
with the compound; and determining the ability of the compound to modulate
The invention also provides methods for modulating neurogenesis. The methods
involve contacting a neural stem or progenitor cell with an effective amount
of a compound that modulates prokineticin receptor signaling. Such methods
are useful in both ex vivo and in vivo therapeutic applications.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the determination that prokineticins
effectively promote neurogenesis in rats. Based on this determination of
this important pharmacological role of prokineticins, the invention provides
screening methods for identifying compounds that modulate neurogenesis and
therapeutic methods for modulating neurogenesis in mammals.
As disclosed in Example V, administration of recombinant human prokineticin
2 (PK2) to adult rats via intracerebroventricular infusion resulted in an
increased number of newly developed neurons in the forebrain. Specifically,
incorporation of the cell proliferation marker BrdU was at least two-fold
higher in the subventricular zone in rats treated with PK2 in comparison to
control animals. Also disclosed herein are the observations that mRNAs for
two related GPCRs, designated prokineticin receptor 1 (PKR1) and
prokineticin receptor 2 (PKR2), are expressed at high levels in the
subventricular zone (SVZ) of the lateral ventricle, the olfactory
bulb/olfactory ventricle, and the dentate gyrus of the hippocampus of adult
mouse brain (see Example I). Further disclosed in Example II is that mRNA
for PKR2 is also expressed in the inner nuclear layer of the adult mouse
retina. mRNA for prokineticin 2 (PK2), which is an agonist of both PKR1 and
PKR2, is expressed in both the olfactory bulb and the dentate gyrus of the
hippocampus of the adult mouse brain. Non-neurogenic surrounding tissues
express PKR1, PKR2 or PK2 at much lower levels. These findings indicate a
role for signaling through prokineticin receptors in the regulation of
Methods for modulating neurogenesis have a variety of important
applications, including for treating individuals having, or who are likely
to develop, disorders relating to neural degeneration, neural damage and
neural demyelination, as described in more detail below. Therapeutic methods
of modulating neurogenesis include both drug-based therapies, in which
compounds that modulate neurogenesis are administered to an individual, and
cell-based therapies, in which neural cells generated, propagated or
genetically modified ex vivo using compounds that modulate neurogenesis are
transplanted into the nervous system of an individual. Developing methods
for modulating neurogenesis ex vivo are also useful for advancing
understanding of the complex processes of neural proliferation and
differentiation, which can lead to development of additional compounds and
approaches for diagnosing and treating disorders of the nervous system.
Accordingly, the invention provides methods of screening for compounds that
modulate neurogenesis. The invention screening methods involve providing a
compound that modulates PKR signaling, and determining the ability of the
compound to modulate neurogenesis.
The invention also provides methods of modulating neurogenesis by contacting
a neural stem or progenitor cell with an effective amount of a compound that
modulates PKR signaling.
A compound that modulates PKR signaling will generally promote signaling in
a suitable signaling assay, as described below, by at least about 10%, such
as at least 25%, 50%, 100%, 500% or more, or alternatively reduce signaling
in a suitable signaling assay by at least about 10%, such as at least 25%,
50%, 90% or more, in comparison to a control compound.
It will be appreciated that PKR signaling can be modulated either directly
or indirectly, and by altering either activity or abundance of either a PKR
receptor or of a PKR ligand. For example, PKR signaling can be modulated
directly, such as by contacting the PKR with an agonist or antagonist. PKR
signaling can also be modulated indirectly, such as by altering PKR
expression, localization, stability, or ability to bind effector molecules,
or by altering prokineticin (PK) expression, secretion, stability or ability
to bind or activate a PKR. Various methods of modulating PKR signaling are
described below, following a description of prokinetin receptors and
prokineticins. Other general approaches to qualitatively or quantitatively
modulate signaling through G-protein coupled receptors are known in the art,
and can be applied in accordance with the invention to prokineticin
receptors given the guidance herein.
As used herein, the term "prokineticin receptor" or "PKR" refers to a
heptahelical membrane-spanning polypeptide that binds to a prokineticin and
signals through a G-protein coupled signal transduction pathway in response
to prokineticin binding. Prokineticin receptors are believed to couple
exclusively to the G.alpha. subtype known as G.alpha.q, and thereby mediate
intracellular calcium mobilization in response to agonists.
A prokineticin receptor can have the naturally-occurring amino acid sequence
of a PKR from any species, or can contain minor modifications with respect
to the naturally-occurring sequence. For example, a PKR can be a mammalian
PKR, such as human PKR1 (SEQ ID NO:1; GenBank Accession No. AAM48127; also
called. GPR73, fb41a, hZAQ, hGPRv21 and EG-VEGF receptor-1; Lin et al., J.
Biol. Chem. 277:19276-19280 (2002), Masuda et al., Biochem. Biophys. Res.
Commun. 293:396-402 (2002), WO 00/34334, WO 01/48188 and WO 01/16309); human
PKR2 (SEQ ID NO:2; GenBank Accession No. AAM48128; also known as I5E, hRUP8
and hZAQ2; Lin et al., supra (2002), Masuda et al., supra (2002), WO
98/46620, WO 01/36471 and WO 02/06483); mouse PKR1 (SEQ ID NO:3; GenBank
Accession No. AAM49570; Cheng et al., Nature 417:405-410 (2002) and WO
02/06483); mouse PKR2 (SEQ ID NO:4; GenBank Accession No. AAM49571; Cheng et
al., supra (2002) and WO 02/06483); rat PKR1 (WO 02/06483); rat PKR2 (WO
02/06483); monkey PKR2 (also known as AXOR8; WO 01/53308); bovine PKR1
(Masuda et al., supra (2002), or a PKR of another mammalian species, such as
other primate, dog, cat, pig, sheep or goat; or a PKR of another vertebrate
species, such as an amphibian, reptile, fish or bird.
Based on the high degree of homology between the nucleotide sequences
encoding PKR1 and PKR2, and between PKR types across species, the skilled
person can readily identify and clone a PKR from any other species. For
example, other PKRs can be identified from nucleic acid libraries using
standard molecular biology approaches such as hybridization and polymerase
chain reaction (PCR) techniques. Additionally, PKRs from other species can
be identified from nucleic acid sequence databases specific for the species
of interest. Methods of identifying, isolating and propagating homologous
nucleic acid molecules are well known in the art and described, for example,
in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold
Spring Harbor Press, Plainview, N.Y. (2001) and Ausubel et al. (Current
Protocols in Molecular Biology, John Wiley & Sons, New York ((current
Likewise, based on the high degree of homology among PKR polypeptides, the
skilled person can readily identify a cell from another species or from an
expression library that expresses a PKR, using PKR-specific antibodies.
Methods of preparing and using antibodies to identify polypeptides are well
known in the art and described, for example, in Harlow and Lane, Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)
A prokineticin receptor can also contain minor modifications with respect to
a naturally-occurring PKR, so long as the PKR retains its ability to bind
to, and signal in response to, a prokineticin, such as a human PK1 or human
PK2 described below.
A prokineticin receptor that contains minor modifications with respect to a
naturally-occurring PKR can contain one or more additions, deletions, or
substitutions of natural or non-natural amino acids relative to the
naturally-occurring polypeptide sequence. Such a modification can be, for
example, a conservative change, wherein a substituted amino acid has similar
structural or chemical properties, for example, substitution of an apolar
amino acid with another apolar amino acid, substitution fo a charged amino
acid with another amino acid of similar charge, and the like. Such a
modification can also be a non-conservative change, wherein a substituted
amino acid has different but sufficiently similar structural or chemical
properties so as to not adversely affect the desired biological activity.
Further, a minor modification can be the substitution of an L-configuration
amino acid with the corresponding D-configuration amino acid with a
non-natural amino acid.
In addition, a minor modification can be a chemical or enzymatic
modification to the polypeptide, such as replacement of hydrogen by an
alkyl, acyl, or amino group; esterification of a carboxyl group with a
suitable alkyl or aryl moiety; alkylation of a hydroxyl group to form an
ether derivative; phosphorylation or dephosphorylation of a serine,
threonine or tyrosine residue; or N- or O-linked glycosylation.
Those skilled in the art can determine whether minor modifications to a
naturally occurring prokineticin receptor sequence is advantageous in a
method of the invention. Minor modifications are useful, for example, to
enhance the stability, selectivity, bioactivity of a receptor. Modified PKR
polypeptides can be prepared, for example, by recombinant methods, by
synthetic methods, by post-synthesis chemical or enzymatic methods, or by a
combination of these methods, and tested for ability to bind a prokineticin
or signal through a G-protein coupled signal transduction pathway.
Those skilled in the art can readily predict regions in a prokineticin
receptor amino acid sequence that can be modified without abolishing binding
or signaling. Such prediction can be based on sequence analysis,
structure-function studies, and computer algorithms. For example,
comparisons of amino acid sequences of PKR2 and PKR1, and of PKRs across
species, can provide guidance in determining amino acid residues that are
tolerant of modification. Further, a large number of published GPCR-structure-function
studies have indicated regions of GPCRs involved in ligand interaction,
G-protein coupling and in forming transmembrane regions, and indicate
regions of GPCRs tolerant of modification (see, for example, Burstein et
al., J. Biol. Chem., 273:24322-24327 (1998) and Burstein et al.,
Biochemistry, 37(12):4052-4058 (1998)). In addition, computer programs known
in the art can be used to determine which amino acid residues of a
polypeptide can be modified without abolishing activity (see, for example,
Eroshkin et al., Comput. Appl. Biosci. 9:491-497 (1993); Gueros et al., J.
Mol. Biol. 320:369-387(2002)). These methods can likewise be applied to PKRs.
For certain applications, a PKR has at least 80%, such as at least 85%, 90%,
95%, 97%, 99% or greater identity with either human PKR1 (SEQ ID NO:1) or
with human PKR2 (SEQ ID NO:2).
That a presumptive PKR, such as a PKR from another species or a modified PKR
is actually a PKR can be confirmed by prokineticin binding assays and
G-protein coupled receptor signaling assays known in the art (see, for
example, U.S. published application 20020115610A1, Lin et al., supra (2002)
and Masuda et al., supra (2002)), and described further below.
Depending on the intended application, the skilled person can determine an
appropriate form for the PKR, such as in a live animal, a tissue, a tissue
extract, a cell, a cell extract, or in substantially purified form. For
example, for receptor binding or signaling assays, the PKR will typically be
either endogenously expressed or recombinantly expressed at the surface of a
Cells that endogenously express a PKR are well known in the art, and
include, for example, M2A7 melanoma cells (available from American Type
Culture Collection as ATCC CRL-2500), M2 melanoma cells (Cunningham et al.,
Science 255;325-327 (1992)) and RC-4B/C pituitary tumor cells (ATCC
CRL-1903)(see U.S. 20020115610A1). Other cells that endogenously express a
PKR include, for example, ileal and other gastrointestinal cells (see U.S.
20020115610A1), endothelial cells such as BACE cells (Masuda et al., supra
(2002)), and endocrine cells (Lin et al., supra (2002)). As disclosed
herein, other cells that express PKR include neural stem and progenitor
cells, including such cells in the subventricular zone of the lateral
ventricle, the olfactory bulb/olfactory ventricle, the dentate gyrus of the
hippocampus, and the inner nuclear layer of the retina.
Methods of recombinantly expressing a PKR are also well known in the art
(see, for example, Lin et al., supra (2002) and Masuda et al., supra
(2002)). Host cells suitable for recombinantly expressing polypeptides are
well known in the art, and include bacterial cells (e.g. E. coli), insect
cells (e.g. Drosophila), yeast cells (e.g. S. cerevisiae, S. pombe, or
Pichia pastoris), and vertebrate cells (e.g. mammalian primary cells and
established cell lines; and amphibian cells, such as Xenopus embryos and
oocytes). Vectors appropriate for expressing polypeptides in the particular
host cell are also well known in the art, and include, for example, vectors
derived from a virus, such as a bacteriophage, a baculovirus or a
retrovirus, and vectors derived from bacteria or a combination of bacterial
sequences and sequences from other organisms, such as a cosmid or a plasmid.
Suitable expression vectors generally contain elements such as an origin of
replication compatible with the intended host cells; constitutive or
inducible promoter sequences; transcription termination and RNA processing
signals; one or more selectable markers compatible with the intended host
cells; one or more multiple cloning sites; and optionally contain tag
sequences that facilitate expression or purification of the encoded
polypeptide. Methods of recombinantly expressing polypeptides are well known
in the art and described, for example, in Sambrook et al., supra (2001) and
Ausubel et al. supra (current supplement).
As used herein, the term "prokineticin" or "PK" refers to a peptide that
binds to a prokineticin receptor and elicits signaling by the receptor
through a G-protein coupled signal transduction pathway.
A prokineticin can have the naturally-occurring amino acid sequence of a PK
from any species, or can contain minor modifications with respect to the
naturally-occurring sequence. For example, a PK can be a mammalian PK, such
as human PK1 (SEQ ID NO:5; GenBank Accession No. P58294; also known as
endocrine-gland-derived endothelial growth factor or EG-VEGF, TANGO 266, PRO
1186 and Zven2; Li et al., supra (2001), LeCouter et al., Nature 412:
877-884 (2001), WO 01/36465, WO 99/63088 and WO 00/52022; human PK2 (GenBank
Accession No. Q9HC23; isoform 1, SEQ ID NO:6, Wechselberger et al., FEBS
Lett. 462:177-181 (1999) or isoform 2, SEQ ID NO:7; also known as Zven1, Li
et al., supra (2001)); mouse PK1 (SEQ ID NO:8; GenBank Accession No.
AAM49573); mouse PK2 (SEQ ID NO:9; GenBank Accession No. AAM49572); rat PK1
(SEQ ID NO:10; GenBank Accession No. AAM09104; Masuda et al., supra (2002));
rat PK2 (SEQ ID NO:11; GenBank Accession No. AAM09105; Masuda et al., supra
(2002)), or a PK of another mammalian species, such as other primate, dog,
cat, pig, cow, sheep or goat.
A PK can alternatively be a PK of another vertebrate species, such as a
snake, frog or toad. For example, a PK can be black mamba PK (SEQ ID NO:12;
GenBank-Accession No. P25687; also known as MIT1; Schweitz et al., FEBS Lett.
461:183-188 (1999)); Bombina variegata frog PK (SEQ ID NO:13; GenBank
Accession No. Q9PW66; also known as Bv8; Mollay et al., Eur. J. Pharmacol.
374:189-196 (1999); Bombina maxima toad PK (SEQ ID NO:14; GenBank Accession
No. AAN03822), or a PK from another vertebrate species, such as an
amphibian, reptile, fish or bird.
A prokineticin can also contain minor modifications with respect to a
naturally-occurring PK, so long as the PK retains its ability to bind to,
and elicit signaling by, a prokineticin receptor, such as a human PKR1 or
PKR2 as described above. Examples of types of suitable minor modifications
have been described above with respect to PKRs, and also apply to PKs. Minor
modifications to a PK can be useful, for example, for applications in which
it is desired to enhance a property of the PK, such as stability,
bioavailability, bioactivity or specificity.
Specific examples of prokineticins that are modified from
naturally-occurring sequences while retaining activity include human
prokineticin chimeras having SEQ ID NO:15 (chimera of PK1 at N-terminus, PK2
at C-terminus) and SEQ ID NO:16 (chimera of PK2 at N-terminus, PK1 at
C-terminus). Chimeras between PK1 and PK2 within species, or between
prokineticins across species, so long as they retain PK activity, are also
As described in U.S. 20020115610A1, prokineticins are not tolerant of
additions, deletions or substitutions at the N-terminal 6 amino acids
conserved across all species (AVITGA), or at any of the 10 conserved
cysteine residues. However, modifications at other internal residues are
tolerated, as are modifications at residues C-terminal to the tenth cysteine.
For certain applications, a PK has at least 40%, such as at least 45%, 50%,
60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or greater identity-with either human
PK1 (SEQ ID NO:5) or with human PK2 (SEQ ID NO:6).
That a presumptive PK, such as a PK from another species or a modified PK is
actually a PK can be confirmed by prokineticin binding assays and G-protein
coupled receptor signaling assays known in the art (see, for example, U.S.
20020115610A1, Lin et al., supra (2002) and Masuda et al., supra (2002)),
and described further below.
Depending on the intended application, the skilled person can determine an
appropriate form for the PK, such as in a live animal, a tissue, a tissue
extract, a cell, a cell extract, or in substantially purified form. A
prokineticin can be substantially purified from any tissue, cell or body
fluid source that endogenously contains PK peptide, or can be produced
recombinantly and then substantially purified. Sources of endogenous PK
include milk (Masuda et al., supra (2002)), venom (Masuda et al., supra
(2002); Mollay et al., Eur. J. Pharmacol. 374:189-196 (1999)), various fetal
and adult tissues, including GI tract, liver, spleen, testis and placenta
(U.S. 20020115610A1) and, as disclosed herein, neural stem and progenitor
cells. Methods of substantially purifying prokineticins from such sources
are described in the afore-mentioned references.
Methods of recombinantly expressing prokineticins are also known in the art
(see U.S. 20020115610A1 and Masuda et al., supra (2001) for examples of
bacterial expression and WO 01/36465 and WO 00/52022 for-examples of
eukaryotic expression). Such methods can involve initially expressing the PK
as a fusion protein, such as a fusion with a glutathione-S-transferase tag,
Fc tag, 6X His tag, myc epitope, or other tag sequences known in the art.
Methods of substantially purifying recombinantly expressed prokineticins,
and for removing optional tag sequences, are also known in the art. For
example, U.S. 20020115610A1 describes conditions for refolding and purifying
recombinantly expressed prokineticins that minimize protein aggregation, and
also describes methods of confirming correct disulfide bond formation.
In one embodiment, the invention screening and therapeutic methods involve
providing a compound that is a prokineticin receptor agonist or antagonist.
A PKR agonist or antagonist can optionally be selective for PKR1 or PKR2, or
alternatively be equally active with respect to both PKR1 and PKR2.
As used herein, the term "prokineticin receptor, agonist" refers to a
compound that promotes or enhances normal G-protein coupled signal
transduction through a PKR. A PKR agonist can act by any agonistic
mechanism, such as by directly binding a PKR at the normal ligand binding
site, thereby promoting receptor signaling. A PKR agonist can also act
indirectly, for example, by potentiating the binding activity of the
endogenous ligand, or by altering the conformation of the PKR so as to
increase its signaling activity.
Examples of PKR agonists include naturally-occurring, chimeric and modified
PK peptides, as described above. PKR agonists can also include
peptidomimetics of such PK peptides. Methods of preparing peptidomimetics
are known in the art and reviewed, for example, in Ripka et al., Curr. Opin.
Chem. Biol. 2:441-452 (1998) and al-Obeidi et al., Mol. Biotechnol.
9:205-223 (1998). Other PKR agonists include compounds identified as such by
the screening assays described below. An exemplary partial agonist is
PK2-insert (insertion of 23 amino acids between exon 2 and exon 3 of PK2)
and an exemplary weak agonist is GIL-PK1 (tripeptide Gly-Ile-Leu added to
the N-terminus of PK1).
As used herein, the term "prokineticin receptor antagonist" refers to a
compound that inhibits or decreases normal G-protein coupled signal
transduction through a PKR. A PKR antagonist can act by any antagonistic
mechanism, such as by directly binding a PKR at the PK binding site, thereby
inhibiting binding between the PKR and its ligand. A PKR antagonist can also
act indirectly, for example, by binding a PK, or by altering the
conformation or state of phosphorylation or glycosylation of a PKR, thereby
affecting its ability to bind or respond to ligand. The term "PKR
antagonist" is also intended to include compounds that act as "inverse
agonists," meaning that they decrease PKR signaling from a baseline amount
of constitutive signaling activity.
Examples of PKR antagonists include peptides with either a single N-terminal
residue addition or a single N-terminal residue deletion with respect to
human PK1 and PK2 (see U.S. 20020115610A1). Peptides having similar
modifications to other prokineticins, or other modifications to the
conserved N-terminal six residues of human or other prokineticins, such as
more extensive additions or deletions and substitutions, can also be PKR
antagonists. An exemplary PKR antagonist is MVITGA.
Other examples of PKR antagonists are antibodies selective for a PKR or a PK.
The term "antibody" is intended to include both polyclonal and monoclonal
antibodies, as well as antigen binding fragments of such-antibodies (e.g.
Fab, F(ab').sub.2, Fd and Fv fragments and the like). In addition, the term
"antibody" is intended to encompass non-naturally occurring antibodies,
including, for example, single chain antibodies, chimeric antibodies,
bifunctional antibodies, CDR-grafted antibodies and humanized antibodies, as
well as antigen-binding fragments thereof.
Methods of preparing and isolating antibodies, including polyclonal and
monoclonal antibodies, using peptide and polypeptide immunogens, are well
known in the art and are described, for example, in Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988).
Non-naturally occurring antibodies can be constructed using solid phase
peptide synthesis, can be produced recombinantly or can be obtained, for
example, by screening combinatorial libraries consisting of variable heavy
chains and variable light chains. Such methods are described, for example,
in Huse et al. Science 246:1275-1281 (1989); Winter and Harris, Immunol.
Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Hilyard et
al., Protein Engineering: A practical approach (IRL Press 1992); and
Borrabeck, Antibody Engineerinq, 2d ed. (Oxford University Press 1995).
A prokineticin receptor agonist will generally have an EC.sub.50 that is no
more than 2-fold, 5-fold, 10-fold, 50-fold, 100-fold or 1000-fold higher or
lower than the EC.sub.50 for human PK1 or PK2 in the particular assay. For
therapeutic applications described below, a prokineticin receptor agonist
preferably has an EC.sub.50, and a prokineticin receptor antagonist
preferably has an IC.sub.50, of less than about 10.sup.-7 M, such as less
than 10.sup.-8 M, and more preferably less than 10.sup.-9 or 10.sub.-10 M.
However, depending on the stability, selectivity and toxicity of the
compound, a prokineticin receptor agonist with a higher EC.sub.50, or a
prokineticin receptor antagonist with a higher IC.sub.50, can also be useful
Signaling assays to identify or confirm the activity of PKR agonists and
antagonists are known in the art. Because PKRs are G.alpha.q-coupled
receptors, signaling assays typically used with other G.alpha.q-coupled
GPCRs can be used to determine PKR signaling activity. G.alpha.q-coupled
GPCRs, when bound to ligand, activate phospholipase C (PLC), which cleaves
the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate the
second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).
These second messengers increase intracellular Ca.sup.2+ concentration and
activate the MAP kinase cascade. The change in activity of PLC, or in
abundance of downstream messengers, is a reflection of GPCR activation.
The specificity of G.alpha. subunits for cell-surface receptors is
determined by the C-terminal five amino acids of the G.alpha.. Thus, if it
is desired to assay a GPCR signaling pathway other than a typical G.alpha.q
pathway, a chimeric G.alpha. containing the five C-terminal residues of
G.alpha.q and the remainder of the protein corresponding to another G.alpha.
can be expressed in a cell such that the PKR is coupled to a different
signaling pathway (see, for example, Conklin et al., Nature 363:274-276
(1993), and Komatsuzaki et al., FEBS Letters 406:165-170 (1995)). For
example, a PKR can be coupled to a G.alpha.s or G.alpha.i, and adenylate
cyclase activation or inhibition assayed by methods known in the art.
Depending on the G.alpha. and the assay system, GPCR signals that can be
determined include, but are not limited to, calcium ion mobilization;
increased or decreased production or liberation of arachidonic acid,
acetylcholine, diacylglycerol, cGMP, cAMP, inositol phosphate and ions;
altered cell membrane potential; GTP hydrolysis; influx or efflux of amino
acids; increased or decreased phosphorylation of intracellular proteins; and
activation of transcription of an endogenous gene or promoter-reporter
construct downstream of any of the above-described second messenger
A variety of cell-based GPCR signaling assays, including assays performed in
bacterial, yeast, baculovirus/insect systems and mammalian cells, are
reviewed, for example, in Tate et al., Trends in Biotecha. 14:426-430
(1996). More recently developed GPCR signaling assays include, for example,
AequoScreen, which is a cellular aequorin-based functional assay that
detects calcium mobilization (LePoul et al., J. Biomol. Screen. 7:57-65
(2002)); MAP kinase reporter assays (Rees et al., J. Biomol. Screen. 6:19-27
(2001); and fluorescence resonance energy transfer (FRET) based PLC
activation assays (van der Wal, J. Biol. Chem. 276:15337-15344 (2001)).
Several examples of PKR signaling assays are described in Lin et al., supra
(2002) and in Masuda et al., supra (2002).
Optionally, a compound can initially be tested to determine whether it binds
a PKR, such compounds being likely to act as PKR agonists or antagonists.
Competitive and non-competitive binding assays for detecting ligand binding
to a receptor are described, for example, in Mellentin-Micelotti et al.,
Anal. Biochem. 272:182-190 (1999); Zuck et al., Proc. Natl. Acad. Sci. USA
96:11122-11127 (1999); and Zhang et al., Anal. Biochem. 268;134-142 (1999).
Examples of PKR binding assays are described in Lin et al., supra (2002) and
in Masuda et al., supra (2002).
For certain applications of the invention methods it will be advantageous to
employ a compound that modulates PKR signaling by a mechanism other than by
acting as a prokineticin receptor agonist or antagonist.
Thus, in one embodiment, a compound that modulates PKR signaling is a
compound that modulates endogenous prokineticin mRNA expression. A compound
that modulates PK expression can be, for example, an expressible nucleic
acid molecule that encodes a PK, or that serves to inhibit PK gene
expression, such as an antisense nucleic acid molecule, a ribozyme or an RNA
interference (RNAi) molecule. Methods of preparing such nucleic acid
molecules and using them in ex vivo and in vivo applications are known in
the art. For example, viral delivery systems are reviewed in Mah et al.,
Clin. Pharmacokinet. 41:901-911; non-viral delivery systems in Li et al.,
Curr. Gene Ther. 1:201-226 (2001); RNAi in Hannon, Nature 418:244-251
(2002); antisense in Galderisi et al., J. Cell Physiol. 181:251-257 (1999)
and ribozymes in Lewin et al., Trends Mol. Med. 7:221-228 (2001)).
A compound that enhances endogenous PK mRNA expression can also be a
compound identified by a screening assay. In an exemplary screening assay,
an animal, tissue or cell that endogenously expresses a PK is contacted with
one or more candidate compounds. PK mRNA can be detected and quantitated
using methods known in the art, such as hybridization with a detectable
probe or primer, or PCR, and the mRNA level compared to a control. Such
methods are described, for example, in Sambrook et al., supra (2001) and
Ausubel et al. supra (current supplement). Alternatively, PK peptide can be
detected and quantitated using methods known in the art, such as by assaying
direct or competitive binding to an antibody or receptor. Such methods are
described, for example, in Harlow and Lane, supra (1988).
Alternatively, a screening assay to identify a compound that enhances
endogenous PK mRNA expression can employ a PK promoter operatively linked to
a nucleic acid molecule encoding a reporter gene, such as .beta.-lactamase,
luciferase, green fluorescent protein or .beta.-galactosidase. In such
assays, a cell transfected with the promoter-reporter construct is contacted
with a candidate compound, and the expression of reporter gene product
detected by methods known in the art which depend on the particular
The nucleotide sequences of PK1 and PK2 promoters are known in the art, and
human and mouse promoter sequences are set forth herein as SEQ ID NOS:17-21.
A smaller portion of a prokineticin promoter sequence can alternatively be
used, such as a region of at least about 100 nucleotides, such as at least
about 200, 250, 300, 500, 1000, 1500, 2000, 2500 or more nucleotides
upstream of the transcriptional start site.
Both the PK1 promoter and the PK2 promoters contain presumptive cyclic-AMP
response elements (CREs), in that they contain the core CRE consensus
sequence CGTCA. In the human PK2 promoter, for example, the presumptive CRE
is about 200 nucleotides upstream of the transcriptional start site. Thus,
it is contemplated that compounds that modulate prokineticin mRNA expression
include compounds known or expected to modulate cyclic-AMP production and/or
MAP kinase activation, such as various growth factors.
In another embodiment, a compound that modulates PKR signaling is a compound
that enhances endogenous prokineticin peptide production or secretion.
Methods of detecting prokineticin production secretion can include
bioassays, binding assays, immunoassays and the like, and can be adapted to
prokineticins based on peptide assays known in the art and the guidance
In a further embodiment, a compound that modulates PKR signaling can be a
compound that modulates PKR mRNA or protein expression. A compound that
modulates PKR expression can be, for example, an expressible nucleic acid
molecule that encodes a PKR, or that serves to inhibit PKR gene expression,
such as an antisense nucleic acid molecule, a ribozyme or an RNA
interference (RNAi) molecule. Methods of preparing such nucleic acid
molecules and using them are known in the art, as described above.
A compound that modulates PKR expression can also be a compound identified
from a screening assay. In an exemplary screening assay, an animal, tissue
or cell that endogenously expresses a PKR is contacted with one or more
candidate compounds. Expression of PKR mRNA or protein can be detected and
the level compared to a control using methods known in the art, such as
methods analogous to those described above for detecting increased
prokineticin mRNA or peptide expression.
The skilled person will appreciate that the above methods to identify
compounds that modulate PKR signaling are exemplary. Alternative methods and
mechanisms for modulating GPCR signaling by modulating abundance or activity
of a PKR or its ligand are known in the art or can be determined based on
the guidance herein.
In the screening assays described above, a candidate compound that is tested
for its ability to modulate PKR signaling can be any type of biological or
chemical molecule. For example, the candidate compound can be a naturally
occurring macromolecule, such as a polypeptide, peptidomimetic, nucleic
acid, carbohydrate, lipid, or any combination thereof. A candidate compound
also can be a partially or completely synthetic derivative, analog or
mimetic of such a macromolecule, or a small organic molecule, such as a
molecule prepared by combinatorial chemistry methods. If desired in a
particular assay format, a candidate compound can be detectably labeled or
attached to a solid support.
Methods for preparing large libraries of compounds, including simple or
complex organic molecules, metal-containing compounds, carbohydrates,
peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic
acids, antibodies, and the like, are well known in the art and are
described, for example, in Huse, U.S. Pat. No. 5,264,563; Francis et al.,
Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol.,
2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med.
Res. Rev. 15:481-496 (1995); and the like. Libraries containing large
numbers of natural and synthetic compounds also can be obtained from
The number of different candidate compounds to test in the methods of the
invention will depend on the application of the method. For example, one or
a small number of candidate compounds can be advantageous in manual
screening procedures, or when it is desired to compare efficacy among
several compounds that are known or predicted to act as PKR modulatory
compounds. However, it will be appreciated that the larger the number of
candidate compounds, the greater the likelihood of identifying a compound
having the desired activity in a screening assay. Additionally, large
numbers of compounds can be processed in high-throughput automated screening
assays. Therefore, the term "one or more candidate compounds" can refer, for
example, to 2 or more, such as 5, 10, 15, 20, 50 or 100 or more different
compounds, such as greater than about 10.sup.3, 10.sup.5 or 10.sup.7
different compounds. Such candidate compounds can be assayed simultaneously,
such as in pools of compounds or in parallel assays, or sequentially.
Assay methods for identifying compounds that modulate PKR signaling
generally involve comparison to a control. One type of a "control" is a
preparation that is treated identically to the test preparation, except the
control is not exposed to the candidate compound. Another type of "control"
is a preparation that is similar to the test preparation, except that the
control preparation does not express the receptor, or has been modified so
as not to respond to a PK. In this situation, the response of the test
preparation to a candidate compound is compared to the response (or lack of
response) of the control preparation to the same compound under
substantially the same reaction conditions.
The methods of the invention involve contacting a neural stem or progenitor
cell with a compound that modulates PKR signaling under conditions in which
the compound can modulate neurogenesis.
As used herein, the term "neurogenesis" refers to the process by which
neural stem and progenitor cells give rise to more differentiated neural
cells. Neurogenesis encompasses proliferation of neural stem and progenitor
cells, differentiation of these cells into new neural cell types, as well as
migration and survival of the new cells. The term is intended to cover
neurogenesis as it occurs during normal development, as well as neural
regeneration that occurs following disease, damage or therapeutic
intervention. Neurogenesis is reviewed, for example, in Okano et al., J.
Neurosci. Res. 69:698-707 (2002).
A number of factors have been shown to modulate neurogenesis in adult
mammals. For example, learning and environment enrichment have been shown to
enhance survival of neural stem and/or progenitor cells, whereas stress has
been shown to diminish proliferation of these cells. In animal models of
epilepsy and stroke, neurogenesis is enhanced. At the biochemical level, a
number of molecules have been shown to influence neurogenesis. For example,
the growth factors EGF, bFGF, VEGF, IGF-1, and the monoamine
neurotransmitters have been shown to stimulate neurogenesis, while high
levels of corticosterone, glutamate, gamma-aminobutyric acid, and opioid
peptides diminish neurogenesis.
As used herein, the term "neural stem cell" refers to a cell of the central
nervous system that can self-replicate and can also give rise to cells of a
plurality of neural cell lineages, such as astrocytes, oligodendrocytes and
As used herein, the term "neural progenitor cell" refers to a cell of the
central nervous system that can proliferate but is more lineage-restricted
in comparison with a neural stem cell. Neural progenitor cells include
neuronal progenitor cells, which produce neurons, and glial progenitor cells
that produce astroglial and/or oligodendroglial cells.
Neurogenesis has been shown to occur throughout adulthood in several
neurogenic areas of the mammalian central nervous system, including the
olfactory bulb (OB), the dentate gyrus (DG) of the hippocampus and the
subventricular zone (SVZ). Low levels of neurogenesis have also been
reported in the Ammon's horn. The new neuronal cells in the adult mammalian
OB are generated from neural progenitor cells in the anterior part of the
SVZ. The SVZ is a narrow zone of tissue in the wall of the lateral ventricle
in the forebrain. The neural progenitor cells of the SVZ migrate to the OB
via the rostro-migratory stream (RMS), where they differentiate into
interneurons of the OB, known as granule cells and periglomerular cells. The
neural progenitor cells of the adult DG are generated in the subgranular
zone (SGZ) of the DG and differentiate into neuronal and glial cells in the
granular layer of the DG. The newly generated neuronal cells extend axons
into the CA3 region of the hippocampus as soon as 4-10 days after mitosis
(reviewed in Taupin and Gage, J. Neurosci. Res. 69:745-749 (2002)).
Neural stem and/or progenitor cells have been isolated from diverse areas of
the adult central nervous system, including the subventricular zone (SVZ),
hippocampus, septum, striatum, cortex, olfactory bulb, the rostral extension
of the SVZ, in different levels of the spinal cord, including cervical,
thoracic, lumbar, and sacral levels. In the spinal cord, neural stem and/or
progenitor cells can be isolated from the periventricular area and the
parenchyma. Neural stem and/or progenitor cells have also been isolated and
cultured from adult postmortem neural tissues (reviewed in Taupin and Gage,
supra (2002)), and are present in the inner nuclear layer of the adult
Neural stem cells express a number of selective immunocytochemical markers,
including Nestin, an intermediate filament protein (Hockfield and McKay, J.
Neurosci. 5:3310-3328 (1985)); Musashil (Msil), an RNA-binding protein (Sakakibara
et al., Dev. Biol. 176:230-242 (1996)); and Soxl, a transcription factor (Pevny
et al., Development 125:1967-1978 (1998)). These markers are also expressed
by neural progenitor cells, albeit at lower levels. Neural stem and
progenitor cells are also characterized by their expression of the
polysialylated form of the neural cell adhesion molecule (PSA-NCAM;
Rousselot et al., J. Comp. Neurol. 351:51-61(1995)) and by their expression
of the fibroblast growth factor (FGF) and epidermal growth factor (EGF)
receptors (Gritti et al., J. Neurosci. 19:3287-3297 (1999)). Other
characteristics of neural stem and progenitor cells are known in the art and
reviewed, for example, in Okano et al., supra (2002).
Compounds that modulate neurogenesis can act by enhancing or reducing neural
stem and/or progenitor cell proliferation, differentiation, survival or
migration. Assays to identify compounds that modulate neurogenesis can be
performed either ex vivo or in vivo, as described below.
A compound that modulates neurogenesis will generally promote neurogenesis
in a suitable neurogenesis assay, as described below, by at least about 10%,
such as at least 25%, 50%, 100%, 500% or more, or alternatively reduce
neurogenesis in such an assay by at least about 10%, such as at least 25%,
50%, 90% or more, in comparison to a control compound.
Modulation of neurogenesis can be evidenced following contacting the cells
with the compound alone, or with the compound in combination with another
neurogenic modulatory factor, such as EGF, FGF, or VEGF. Modulation of
neurogenesis can also be evidenced under normal ex vivo or in vivo
conditions, or under conditions that mimic neural disease or damage.
Neural stem and/or progenitor cells suitable for use in the assay methods
described below can be obtained from mammals, including humans (post-mortem
or following surgery) and experimental animals (such as rodents, non-human
primates, dogs, cats and the like). Neural stem and/or progenitor cells can
also be obtained from other vertebrates, including reptiles, amphibians,
fish and birds, or from invertebrates. The human or animal can be male or
female, can be fetal, young, adult or old, and can be normal or exhibiting
or susceptible to a neural disease or disorder.
For certain applications, tissue explants can be used, whereas for others,
dissociated cells or neurospheres can be obtained. Tissue explants or cells
can be obtained from any neural tissue that contains neural stem or
progenitor cells, including brain and spinal tissues as described above.
Exemplary tissues include olfactory bulb (OB), the dentate gyrus (DG) of the
hippocampus and the subventricular zone (SVZ) of the lateral ventricles.
Example III describes a method of obtaining neural stem and/or progenitor
cells from the lateral ventricles of adult mice. Other methods are known in
the art (see, for example, U.S. Pat. No. 5,753,506; and Gritti et al., J.
Neurosci. 16:1091-1100 (1996)).
In addition to primary cells, neural stem and/or progenitor cell lines can
be used in neurogenesis assays, such as MHP36 cells of mouse hippocampal
origin (Gray et al., Philos. Trans. Royal Soc. Lond. B. Biol. Sci.
354:1407-1421 (1999)), CSM14.1 cells of rat mesencephalic origin (Haas et
al., J. Anat. 201:61-69 (2002)), and embryonic stem cells differentiated
along the neural lineage.
In ex vivo neurogenesis assays, either proliferation or differentiation, or
both, can be readily assessed by methods known in the art. Proliferation and
differentiation assays can also indirectly measure cell survival.
In an exemplary proliferation assay, a cell composition containing neural
stem and/or progenitor cells can be cultured under conditions in which
clonal spheroid colonies, or "neurospheres," form. These neurospheres can be
visualized and counted under a light microscope. Neurospheres can then be
dissociated, and the culture continued, such that the number of secondary
and subsequent neurospheres can be determined as an indication of
proliferation. Conditions for culturing and dissociating neurospheres are
described in Example III.
In other exemplary proliferation assays, the cells are assessed for their
ability to incorporate .sup.3H thymidine or bromodeoxyurine (BrdU, a
thymidine analog), or assessed for their expression of proliferation
markers, such as proliferating cell nuclear antigen (PCNA) or cdc2. Such
proliferation assays are well known in the art and are described, for
example, in Freshney, "Culture of Animal Cells: A Manual of Basic Technique"
New York: Wiley-Liss, 4.sup.th ed. (2000).
Other types of proliferation assays suitable for determining whether a
compound modulates neurogenesis are known in the art or can be determined
using the guidance herein. The total number of cells, or number of labeled
cells, observed in the presence of compound and in the absence of compound
can be compared to determine if neurogenesis is modulated.
In an exemplary differentiation assay, cells are contacted with suitable
primary antibodies specific for markers expressed by the type or types of
cells it is desired to detect. Markers expressed by neural-stem and
progenitor cells have been described above. As described in Example III, a
marker for neurons is .beta.-tubulin, a marker for astrocytes is GFAP, and a
marker for oligodendrocytes is O4. Other markers specific for cells of
various neural lineages are known in the art, and antibodies thereto are
commercially available or can be generated by known methods.
Unless the primary antibody is labeled, the cells are then generally
contacted with labeled secondary antibodies, such as enzymatically or
fluorescently labeled antibodies, and the cells visualized or sorted.
Methods to immunolabel cells, as well as methods to detect and sort
immunolabeled cells, are well known in the art and described, for example,
in Freshney, supra (2000). The types and numbers of cells of each lineage of
interest observed in the presence of compound and in the absence of compound
can be compared to determine if neurogenesis is modulated.
Neurogenesis assays can be performed in vivo in a human or other mammal,
vertebrate or invertebrate, as described above. Such assays are generally
similar to ex vivo assays, except that the tissue of interest is generally
fixed and sectioned prior to detection of the label or marker of interest.
Example IV describes a method of labeling proliferating cells in vivo with
BrdU and subsequently detecting BrdU-labeled cells, and cells expressing
neural stage-specific differentiation markers, by immunohistochemistry.
Alternatively, and particularly where the human or animal is not sacrificed,
detection of proliferation and differentiation markers can be done using
radiolabeled antibodies and non-invasive imaging methods known in the art,
such as single photon emission computed tomography (SPECT) and positron
emission tomography (PET).
For in vivo applications, various delivery methods can be used to contact
neural stem or progenitors within the tissue of interest with a compound, as
described in more detail below with respect to therapeutic applications. The
delivery method will depend on factors such as the tissue of interest, the
nature of the compound (i.e. its stability and ability to cross the
blood-brain barrier), and the duration of the experiment. As described in
Example IV, for delivery of peptides and BrdU to the brain over a period of
several weeks, an osmotic minipump can be implanted into a neurogenic
region, such as the lateral ventricle. Alternatively, compounds can be
administered by direct injection into the cerebrospinal fluid of the brain
or spinal column, or into the eye. Compounds can also be administered into
the periphery (such as by intravenous or subcutaneous injection, or oral
delivery), and subsequently cross the blood-brain barrier.
Advantageously, in vivo methods allow the effect of compounds to be tested
for their effect on neurogenesis both in normal subjects and in subjects
having neural damage and disease. Either human subjects or experimental
animal models can be used.
Experimental animal models of trauma due to stroke or neural injury are
known in the art. One experimental model of stroke involves occluding the
right middle cerebral artery and both common carotid arteries of rats for a
short period, followed by reperfusion (Moore et al., J. Neurochem.
80:111-118). An experimenal model of CNS injury is the fluid percussion
injury (FPI) model, in which moderate impact (1.5-2.0 atm) is applied to the
parietal cerebral cortex (Akasu et al., Neurosci. Lett. 329:305-308 (2002).
Experimental models of spinal cord injury are also used in the art (Scheifer
et al., Neurosci. Lett. 323:117-120 (2002). Suitable models for neural
damage due to oxidative stress, hypoxia, radiation and toxins are also known
in the art.
Experimental animal models of human neurodegenerative diseases are also
known in the art. Various experimental models of Alzheimer's disease are
reviewed in Janus, Physiol. Behav. 73:873-886 (2001); models of Parkinson's
disease are reviewed in Tolwani et al., Lab. Anim. Sci. 49:363-371 (1999);
models of Huntington's disease are reviewed in Menalled et al., Trends
Pharmacol. Sci. 23:32-39 (2002); and a SOD-1 transgenic model of amyotrophic
lateral sclerosis is described in Ripps et al., Proc. Natl. Acad. Sci. USA
92:689-693 (1995). Other animal models for human neural degenerative
diseases, including those described below with respect to therapeutic
applications, are known in the art.
Experimental animal models of retinal neurogenesis are described, for
example, in Marcus et al., Visual Neurosci. 16:417-424 (1999).
Experimental animal models of demyelinating diseases, such as experimental
autoimmune encephalomyelitis (a model of multiple sclerosis), are also known
in the art.
In any of the in vivo assays, neurogenesis in the presence of compound and
in the absence of compound can be compared to determine if the compound
modulates neurogenesis. Such in vivo assays can further provide evidence of
safety, toxicity, pharmacokinetics and therapeutic efficacy of the compound
of interest in preparation for human therapeutic use.
The compounds of the invention that modulate neurogenesis can be used
directly as therapeutic agents to prevent or treat a variety of disorders of
the nervous system in which it is beneficial to promote or inhibit
neurogenesis. The compounds of the invention can also be used to promote
neurogenesis ex vivo, such that a cell composition containing neural stem
cells, neural progenitor cells, and/or more differentiated neural cells can
subsequently be administered to an individual to prevent or treat the same
Nervous system disorders that can be treated with the compounds of the
invention include, but are not limited to, nervous system injuries, and
diseases or disorders which result in either a disconnection of axons, a
diminution or degeneration of neurons, or demyelination. Such diseases and
disorders include, for example, the following lesions of the central nervous
system (including spinal cord and brain) or peripheral nervous systems: (1)
ischemic lesions, in which a lack of oxygen in a portion of the nervous
system results in neuronal injury or death, including cerebral infarction or
ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions,
including lesions caused by physical injury or associated with surgery, for
example, lesions which sever a portion of the nervous system, or compression
injuries; (3) malignant lesions, in which a portion of the nervous system is
destroyed or injured by malignant tissue which is either a nervous system
associated malignancy or a malignancy derived from non-nervous system
tissue; (4) infectious lesions, in which a portion of the nervous system is
destroyed or injured as a result of infection, for example, by an abscess or
associated with infection by human immunodeficiency virus, herpes zoster, or
herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5)
degenerative lesions, in which a portion of the nervous system is destroyed
or injured as a result of a degenerative process including but not limited
to, degeneration associated with Parkinson's disease, Alzheimer's disease,
Huntington's chorea, amyotrophic lateral sclerosis (ALS) and retinal
degeneration; (6) lesions associated with nutritional diseases or disorders,
in which a portion of the nervous system is destroyed or injured by a
nutritional disorder or disorder of metabolism including, but not limited
to, vitamin B12 deficiency, folic acid deficiency, Wemicke disease,
tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration
of the corpus callosum), and alcoholic cerebellar degeneration; (7)
neurological lesions associated with systemic diseases including, but not
limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus
erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic
substances including alcohol, lead, or particular neurotoxins; and (9)
demyelinated lesions in which a portion of the nervous system is destroyed
or injured by a demyelinating disease including, but not limited to,
multiple sclerosis, human immunodeficiency virus-associated myelopathy,
transverse myelopathy or various etiologies, progressive multifocal
leukoencephalopathy, and central pontine myelinolysis.
Because neurogenesis is involved in learning and memory, compounds of the
invention can also be used in normal individuals to enhance learning and/or
memory, or to treat individuals with defects in learning and/or memory.
Other conditions that can be beneficially treated with compounds that
modulate neurogenesis are known in the art (see, for example, U.S. published
It is expected that the compounds identified by the methods described herein
as compounds that modulate PKR signaling and further modulate neurogenesis
will have beneficial activities in addition to modulating neurogenesis. For
example, PKR1 and/or PKR2 are expressed on a variety of tissues apart from
the neural tissues described herein. Modulation of PKR signaling has been
proposed to be beneficial to treat conditions relating to these tissues,
such as disorders of gastrointestinal motility (see U.S. 20020115610A1),
circadian rhythm (Cheng et al., Nature 417:405-410 (2002)) and angiogenesis
(LeCouter et al., supra (2001)).
Stem cells have the ability to divide for indefinite periods in culture and
to give rise to specialized cells. The fertilized egg is totipotent, meaning
that its potential is total. After several cycles of cell division, these
totipotent cells begin to specialize, forming a hollow sphere of cells,
called a blastocyst. The cells of the inner cell mass of the blastocyst are
pluripotent, and will go on to form many but not all types of cells
necessary for fetal development. The pluripotent stem cells undergo further
specialization into stem cells that are committed to give rise to cells that
have a particular function. These cells are called multipotent stem cells.
Examples of multipotent stem cells include hematopoietic stem cells, which
give rise to red blood cells, white blood cells and platelets; hepatic stem
cells, which differentiate into hepatocytes and biliary epithelial cells;
myogenic stem cells, which differentiate into muscle; and neural stem cells
described above. Multipotent stem cells have been identified in many other
organs and tissues, including lung, heart, prostate, skin and
Because neural stem cells share certain features with other types of stem
cells, such as certain surface markers (see, for example, Klassen et al.,
Neurosci. Lett. 312:180-182 (2001); Hunziker et al., Biochem. Biophys. Res.
Commun. 271:116-119 (2000)), as well as the ability to self-replicate and
differentiate, it is expected that signaling through prokineticin receptors,
disclosed herein to be implicated in neurogenesis, is also important in
modulating proliferation and differentiation of pluripotent and other
multipotent stem cells. Accordingly, the methods described herein for
identifying and using compounds that modulate neurogenesis can be readily
applied to identifying and using compounds that modulate proliferation and
differentiation of other stem cells in ex vivo and in vivo applications
known in the art.
Those skilled in the art can determine other useful applications for
compounds that modulate PKR signaling.
Compounds of the invention that modulate neurogenesis can be formulated and
administered in a manner and in an amount appropriate for the condition to
be treated; the weight, gender, age and health of the individual; the
biochemical nature, bioactivity, bioavailability and side effects of the
particular compound; and in a manner compatible with concurrent treatment
regimens. An appropriate amount and formulation for a particular therapeutic
application in humans can be extrapolated based on the activity of the
compound in the ex vivo and in vivo neurogenesis assays described herein.
The total amount of a compound can be administered as a single dose or by
infusion over a relatively short period of time, or can be administered in
multiple doses administered over a more prolonged period of time.
Additionally, the compound can be administered in a slow-release matrice,
which can be implanted for systemic delivery at or near the site of the
target tissue. Contemplated matrices useful for controlled release of
compounds, including therapeutic compounds, are well known in the art, and
include materials such as DepoFoam.TM., biopolymers, micropumps, and the
The invention compounds can be administered to a mammal by a variety of
routes known in the art including, for example, intracerebrally,
intraspinally, intravenously, intramuscularly, subcutaneously,
intraorbitally, intracapsularly, intraperitoneally, intracisternally, intra-articularly,
orally, intravaginally, rectally, topically, intranasally, or transdermally.
Generally, the invention compounds are administered to an animal as a
pharmaceutical composition comprising the compound and a pharmaceutically
acceptable carrier. The choice of pharmaceutically acceptable carrier
depends on the route of administration of the compound and on its particular
physical and chemical characteristics. Pharmaceutically acceptable carriers
are well known in the art and include sterile aqueous solvents such as
physiologically buffered saline, and other solvents or vehicles such as
glycols, glycerol, oils such as olive oil and injectable organic esters. A
pharmaceutically acceptable carrier can further contain physiologically
acceptable compounds that stabilize the compound, increase its solubility,
or increase its absorption. Such physiologically acceptable compounds
include carbohydrates such as glucose, sucrose or detrains; antioxidants,
such as ascorbic acid or glutathione; chelating agents; and low molecular
weight proteins (see for example, "Remington's Pharmaceutical Sciences" 18th
ed., Mack Publishing Co. (1990)).
For applications that require the compounds to cross the blood-brain
barrier, or to cross cell membranes, formulations that increase the
lipophilicity of the compound can be useful. For example, the compounds of
the invention can be incorporated into liposomes (Gregoriadis, Liposome
Technoloqy, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)).
Liposomes, which consist of phospholipids or other lipids, are nontoxic,
physiologically acceptable and metabolizable carriers that are relatively
simple to make and administer. Other approaches for formulating a compound
such that it crosses the blood-brain barrier are known in the art and
include the use of nanoparticles, which are solid colloidal particles
ranging in size from 1 to 1000 nm (Lockman et al., Drug Dev. Ind. Pharm.
28:1-13 (2002)), and peptides and peptidomimetics that serve as transport
vectors (Pardridge, Nat. Rev. Drug Discov. 1:131-139 (2002).
Although drug delivery to the central nervous system poses unique
challenges, the CNS is also a site of immune privilege. Thus, a compound
that could elicit an immune response that precludes its use for many
indications can still be an effective therapeutic if delivered into the CNS.
Accordingly, compounds that are potentially immunogenic, such as chimeric
prokineticin peptides, prokineticins from other species, and certain other
PKR modulatory compounds, can be used as effective therapeutics to modulate
neurogenesis in humans.
Besides peptides and compounds identified in drug screens, PKR modulatory
compounds described herein include nucleic acid molecules that encode
prokineticins or prokineticin receptors, as well as ribozymes, antisense
molecules, RNAi and the like. Methods of formulating and delivering nucleic
acid molecules to individuals for therapeutic use are known in the art.
Useful vectors for delivering therapeutic nucleic acid molecules to the CNS
include, for example, herpes simplex virus vectors, adenoviral vectors,
VSV-G-pseudotyped retroviral vectors and adeno-associated viral vectors.
Methods for delivering nucleic acid molecules to the CNS are reviewed, for
example, in Hsich et al., Hum. Gene Ther. 13:579-604 (2002).
Cells that have been contacted with PKR modulatory compounds, including
neural stem and progenitor cells, more differentiated cells, and genetically
modified cells expressing PK, can be used in therapeutic applications
described herein. Methods of formulating and delivering cells to individuals
for therapeutic use are known in the art (see, for example, reviews by Cao
et al., J. Neurosci. Res. 68:501-510 (2002), and Park et al., Gene Ther.
To enhance the modulation of neurogenesis, more than one therapeutic
approach or composition can be provided to an individual or used ex vivo.
For example, a compound that modulates PKR signaling can be used in
conjunction with neurogenic growth factors, such as EGF and FGF, or in
conjunction with conventional therapies for the disorder or condition being
treated. The skilled clinician will be able to determine appropriate
concurrent or sequential therapies for use in conjunction with the
compounds-and methods of-the invention.
As described herein, PKR1, PKR2 and PK2 are expressed on cells within
regions of the brain and retina that are neurogenically active. Accordingly,
molecules that specifically bind these polypeptides can be used in either ex
vivo or in vivo applications to label neurogenically active cells or
regions. Molecules that specifically bind PKRs and PKs include their cognate
ligands and receptors, respectively, as well as antibodies and binding
compounds identified in the assays described herein.
Thus, the invention provides a method of labeling neural cells, by
contacting a mixed cell population, tissue or animal with a ligand that
binds a PKR or a PK, and detecting binding of said ligand to a cell within
said population, tissue or animal.
The use of prokineticins and prokineticin receptors as markers is
particularly advantageous in cases in which these markers are more abundant
or more selective for particular cell types than other markers currently
used to identify neural cells, such as the markers described above. The
labeling method can be used in a variety of research and clinical
applications, such as in identifying and distinguishing different types of
cells, in diagnosing neural disorders, and in monitoring the efficacy of
therapies with neurogenic agents. The skilled person can determine other
useful applications for the compounds and methods described herein.
Claim 1 of 9 Claims
1. A method for modulating neurogenesis,
comprising contacting a neural stem or progenitor cell with an effective
amount of a prokineticin receptor (PKR) agonist compound wherein the PKR
agonist is selected from the group consisting of SEQ ID NOS:5-11 and
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