U.S. patent application number 09/920085 was filed with the patent office on 2002-01-10 for methods for treating neurological deficits.
This patent application is currently assigned to The Regents of the University of California, a California corporation. Invention is credited to Fallon, James H., Reid, James Steven.
Application Number | 20020004039 09/920085 |
Document ID | / |
Family ID | 21997438 |
Filed Date | 2002-01-10 |
United States Patent
Application |
20020004039 |
Kind Code |
A1 |
Reid, James Steven ; et
al. |
January 10, 2002 |
Methods for treating neurological deficits
Abstract
The present invention features methods and compositions for
treating a patient who has a neurological deficit. The method can
be carried out, for example, by contacting (in vivo or in culture)
a neural progenitor cell of the patient's central nervous system
(CNS) with a polypeptide that binds the epidermal growth factor
(EGF) receptor and directing progeny of the proliferating
progenitor cells to migrate en masse to a region of the CNS in
which they will reside and function in a manner sufficient to
reduce the neurological deficit. The method may include a further
step in which the progeny of the neural precursor cells are
contacted with a compound that stimulates differentiation.
Inventors: |
Reid, James Steven;
(Berkeley, CA) ; Fallon, James H.; (Irvine,
CA) |
Correspondence
Address: |
MICHAEL P. REED, PH.D.
Fish & Richardson P.C.
Suite 500
4350 La Jolla Village Drive
San Diego
CA
92122
US
|
Assignee: |
The Regents of the University of
California, a California corporation
|
Family ID: |
21997438 |
Appl. No.: |
09/920085 |
Filed: |
July 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09920085 |
Jul 31, 2001 |
|
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09129028 |
Aug 4, 1998 |
|
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60055383 |
Aug 4, 1997 |
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Current U.S.
Class: |
424/93.7 ;
435/368 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 25/28 20180101; A61K 38/185 20130101; A61P 25/00 20180101;
A61K 38/1841 20130101; A61P 27/02 20180101 |
Class at
Publication: |
424/93.7 ;
435/368 |
International
Class: |
A61K 045/00; C12N
005/08 |
Claims
What is claimed is:
1. A method for treating a patient who has a neurological deficit,
the method comprising (a) contacting a neural progenitor cell of
the patient's central nervous system (CNS) with a polypeptide that
binds the epidermal growth factor (EGF) receptor, the dosage of the
polypeptide being sufficient to stimulate the proliferation of the
neural progenitor cell, and (b) directing progeny of the
proliferating progenitor cell to migrate en masse to a region of
the CNS in which the cells will reside and function in a manner
sufficient to reduce the neurological deficit.
2. The method of claim 1, further comprising contacting the cells
with a compound that stimulates the progeny of the proliferating
neural progenitor cells to differentiate.
3. The method of claim 1, wherein the neurological deficit is
caused by a neurodegenerative disease, a traumatic injury, a
neurotoxic injury, ischemia, a developmental disorder, a disorder
affecting vision, an injury or disease of the spinal cord, a
demyelinating disease, an autoimmune disease, an infection, or an
inflammatory disease.
4. The method of claim 3, wherein the neurodegenerative disease is
Alzheimer's Disease, Huntington's Disease, or Parkinson's
Disease.
5. The method of claim 3, wherein the ischemia is associated with a
stroke.
6. The method of claim 1, wherein the polypeptide that binds the
EGF receptor is amphiregulin (AR), betacellulin (BTC), epidermal
growth factor (EGF), epiregulin (ER), heparin-binding EGF-like
growth factor (HB-EGF), schwannoma-derived growth factor (SDGF),
myxomavirus growth factor Shope fibroma virus growth factor,
teratocarcinoma-derived growth factor-1 (TDGF-1), transforming
growth factor alpha (TGF.alpha.), or vaccinia growth factor
(VGF).
7. The method of claim 1, wherein the polypeptide that binds the
EGF receptor is TGF.alpha..
8. The method of claim 1, wherein the neural progenitor cell is
contacted in vivo with a polypeptide that binds the EGF
receptor.
9. The method of claim 1, wherein the neural progenitor cell is
contacted in culture with a polypeptide that binds the EGF
receptor.
10. The method of claim 1, wherein migration is directed by
contacting a cell along, or at the end of, a desired path of
migration with a compound that increases the expression of a cell
adhesion molecule or extracellular matrix molecule.
11. The method of claim 10, wherein the compound is TGF.alpha..
12. The method of claim 10, wherein the cell adhesion molecule is
fibronectin.
13. The method of claim 10, wherein the cell adhesion molecule is
laminin.
14. The method of claim 10, wherein the compound is applied along
the path between the neural precursor cells and the location to
which their progeny are directed to migrate.
15. The method of claim 1, wherein migration is directed by
contacting the cells along a desired migratory path with a compound
that inhibits a naturally occurring signal along the path, the
naturally occurring signal being a signal that inhibits
migration.
16. The method of claim 1, wherein migration is directed by
mechanically disrupting tissue in the CNS.
17. The method of claim 1, wherein migration is directed by
neurochemically blocking the activity of cells in the CNS.
18. The method of claim 2, wherein the compound that stimulates
differentiation is retinoic acid or brain-derived neurotrophic
factor.
19. A method for treating a patient who has a neurological deficit,
the method comprising (a) contacting a neural progenitor cell of
the patient's central nervous system (CNS) with a polypeptide that
binds the epidermal growth factor (EGF) receptor, the dosage of the
polypeptide being sufficient to stimulate the proliferation of the
neural progenitor cell; (b) directing the progeny of the
proliferating progenitor cells to migrate en masse to a second
region of the CNS; and (c) contacting the cells that have migrated
with a compound that stimulates differentiation.
20. The method of claim 19, wherein the compound that stimulates
the proliferation of neural stem cells and the compound that
stimulates differentiation are administered sequentially.
21. A pharmaceutical composition comprising a polypeptide that
binds the epidermal growth factor (EGF) receptor and a compound
that stimulates the differentiation of neural progenitor cells.
22. The pharmaceutical composition of claim 21, wherein the
polypeptide that binds the EGF receptor is TGF.alpha..
23. The pharmaceutical composition of claim 21, wherein the
polypeptide that binds the EGF receptor is TGF.alpha. and the
compound that stimulates the differentiation of neural progenitor
cells is brain-derived neurotrophic factor.
24. The method of claim 1, wherein the injury to the central
nervous system is an injury to the spinal cord.
25. The method of claim 1, wherein the injury to the central
nervous system is an injury to the retina.
26. A method for treating a subject having a neurological deficit,
the method comprising contacting a neural precursor cell in vivo
with a therapeutically effective amount of a polypeptide that binds
the epidermal growth factor (EGF) receptor, wherein the polypeptide
is parentally administered to the subject, and wherein the
administration induces the proliferation, migration, or
differentiation of a neural precursor cell in a manner sufficient
to treat the neurological deficit.
27. The method of claim 26, wherein the neurological deficit is
caused by a neurodegenerative disease, a traumatic injury, a
neurotoxic injury, ischemia, a developmental disorder, a disorder
affecting vision, an injury or disease of the spinal cord, a
demyelinating disease, an autoimmune disease, an infection, or an
inflammatory disease.
28. The method of claim 27, wherein the neurodegenerative disease
is Alzheimer's Disease, Huntington's Disease, or Parkinson's
Disease.
29. The method of claim 27, wherein the ischemia is associated with
a stroke.
30. The method of claim 26, wherein the polypeptide that binds the
EGF receptor is amphiregulin (AR), betacellulin (BTC), epidermal
growth factor (EGF), epiregulin (ER), heparin-binding EGF-like
growth factor (HB-EGF), schwannoma-derived growth factor (SDGF),
myxomavirus growth factor Shope fibroma virus growth factor,
teratocarcinoma-derived growth factor-1 (TDGF-1), transforming
growth factor alpha (TGF.alpha.), or vaccinia growth factor
(VGF).
31. The method of claim 26, wherein the polypeptide that binds the
EGF receptor is TGF.alpha..
32. The method of claim 26, wherein migration is directed by
contacting a cell along, or at the end of, a desired path of
migration with a compound that increases the expression of a cell
adhesion molecule or extracellular matrix molecule.
33. The method of claim 32, wherein the compound is TGF.beta..
34. The method of claim 32, wherein the cell adhesion molecule is
fibronectin.
35. The method of claim 32, wherein the cell adhesion molecule is
laminin.
36. The method of claim 32, wherein the compound is applied along
the path between the neural precursor cells and the location to
which their progeny are directed to migrate.
37. The method of claim 26, wherein migration is directed by
contacting the cells along a desired migratory path with a compound
that inhibits a naturally occurring signal along the path, the
naturally occurring signal being a signal that inhibits
migration.
38. The method of claim 26, wherein migration is directed by
mechanically disrupting tissue in the CNS.
39. The method of claim 26, wherein migration is directed by
neurochemically blocking the activity of cells in the CNS.
40. The method of claim 26, wherein differentiation is stimulated
by retinoic acid or brain-derived neurotrophic factor.
41. A method for treating a subject having a neurological deficit,
the method comprising (a) contacting a neural precursor cell in
vivo with a therapeutically effective amount of a polypeptide that
binds the epidermal growth factor (EGF) receptor, wherein the
polypeptide is parenterally administered in a dosage sufficient to
stimulate the proliferation of the neural progenitor cell; (b)
directing the progeny of the proliferating progenitor cells to
migrate en masse to a second region of the CNS; and (c) contacting
the cells that have migrated with a compound that stimulates
differentiation.
42. The method of claim 41, wherein the polypeptide that stimulates
the proliferation of neural stem cells and the compound that
stimulates differentiation are administered sequentially.
43. A pharmaceutical composition comprising a polypeptide that
binds the epidermal growth factor (EGF) receptor and a compound
that stimulates the differentiation of neural precursor cells.
44. The pharmaceutical composition of claim 43, wherein the
polypeptide that binds the EGF receptor is TGF.alpha..
45. The pharmaceutical composition of claim 43, wherein the
polypeptide that binds the EGF receptor is TGF.alpha. and the
compound that stimulates the differentiation of neural precursor
cells is brain-derived neurotrophic factor.
46. The method of claim 26, wherein the injury to the central
nervous system is an injury to the spinal cord.
47. The method of claim 26, wherein the injury to the central
nervous system is an injury to the retina.
Description
[0001] This application claims benefit from provisional application
Ser. No. 60/055,383, filed Aug. 4, 1997, which is hereby
incorporated by reference in its entirety.
[0002] The field of the invention is treatment of neurological
deficits caused by an injury, disease, or developmental disorder
that affects the central nervous system.
BACKGROUND OF THE INVENTION
[0003] Neurotrophic factors are peptides that variously support the
survival, proliferation, differentiation, size, and function of
nerve cells (for review, see Loughlin and Fallon, Neurotrophic
Factors, Academic Press, San Diego, Calif., 1993). While the
numbers of identified trophic factors, or growth factors, are
ever-increasing, most can be assigned to one or another established
family based upon their structure or binding affinities. Growth
factors from various families, including the epidermal growth
factor (EGF) family, have been demonstrated to support dopaminergic
neurons of the nigrostriatal system (an area that can be treated
according to the methods of the present invention) (for review, see
Hefti, J. Neurobiol. 25:1418-1435, 1994; Unsicker, Prog. Growth
Factor Res. 5:73-87, 1994).
[0004] EGF, the founding member of the EGF family, was
characterized more than 25 years, ago (Savage and Cohen, J. Biol.
Chem. 247:7609-7611, 1972; Savage et al., J. Biol. Chem.
247:7612-7621, 1972). Since then, additional members have been
identified; they include vaccinia virus growth factor.(VGF;
Ventatesan et al., J. Virol. 44:637-646, 1982), myxomavirus growth
factor (MGF; Upton et al., J. Virol. 61:1271-1275, 1987), Shope
fibroma virus growth factor (SFGF; Chang et al., Mol. Cell. Biol.
7:535-540, 1987), amphiregulin (A R; Kimura et al., Nature
348:257-260, 1990), and heparin-binding EGF-like growth factor
(HB-EGF; Higashiyama et al., Science 251:936-939, 1991). A common
feature of these factors is an amino acid sequence containing six
cysteines that form three disulfide cross links and support a
conserved structure that underlies their common ability to bind the
EGF receptor.
[0005] EGF is by far the most-studied member of the family and was
the first localized to brain tissue: EGF-like immunoreactivity (IR)
was found in areas of developing adult forebrain and midbrain
including the globus pallidus, ventral pallidum, entopeduncular
nucleus, substantia nigra, and the Islands of Calleja (Fallon et
al., Science 224:1107-1109, 1984).
[0006] Another member of the EGF family, TGF.alpha., has also been
localized to brain tissue. It binds the EGF receptor (Todaro et
al., Proc. Natl. Acad. Sci. USA 77:5258-5262, 1980), stimulates the
receptor's tyrosine kinase activity, and elicits similar mitogenic
responses in a wide variety of cell types (for review, see Derynck,
Adv. Cancer Res. 58:27-52, 1992). TGF.alpha. might also bind to
additional, unidentified receptors (which may help explain its
differential actions in some cells). TGF.alpha.-IR has previously
been shown to be heterogeneously distributed in neuronal perikarya
throughout the adult rat CNS and in a subpopulation of forebrain
astrocytes (Code et al., Brain Res. 421:401-405, 1987; Fallon et
al., Growth Factors 2:241-250, 1990). TGF.alpha. mRNA has been
detected in whole rodent brain (Lee et al., Mol. Cell. Biol.
5:3655-3646, 1985; Kudlow et al., J. Biol. Chem. 264:3880-3883,
1989) and in striatum and other brain regions by a nuclease
protection assay (Weickert and Blum, Devel. Brain Res. 86:203-216,
1995) and by in situ nucleic acid hybridization (Seroogy et al.,
Neuroreport 6:105-108, 1994).
[0007] TGF.alpha. and EGF mRNAs reach their highest relative
abundance (compared to total RNA) in the early postnatal period and
decrease thereafter, suggesting a role in development (Lee et al.,
1985, supra; Lazar and Blum, J. Neurosci. 12:1688-1697, 1992). In
whole brain, the reduction is over 50% (Lazar and Blum, 1992,
supra), whereas, in striatum, relative TGF.alpha. mRNA drops by
two-thirds from peak levels (Weickert and Blum, 1995, supra). At
all developmental stages examined, whole brain TGF.alpha. mRNA
exceeds EGF mRNA levels by more than an order of magnitude (Lazar
and Blum, 1992, supra).
[0008] The EGF receptor was localized by immunocytochemistry to
astrocytes and subpopulations of cortical and cerebellar neurons in
rat brain and to neurons in human autopsy brain specimens
(Gomez-Pinilla et al., Brain Res. 438:385-390, 1988; Werner et al.,
J. Histochem. Cytochem. 36:81-86, 1988). EGF binding sites were
revealed in rat cortical and subcortical areas, including the
striatum, in an autoradiography study with radiolabeled EGF
(Quirion et al., Synapse 2:212-218, 1988). In situ hybridization
studies demonstrated EGF receptor mRNA in striatum and cells of the
ventral mesencephalon (Seroogy et al., 1994, supra) and in
proliferative regions in developing and adult rat brain (Seroogy et
al., Brain Res. 670:157-164, 1995). As with relative EGF and
TGF.alpha. mRNAs, EGF receptor mRNA is most abundant in striatum
and ventral midbrain early in development, and gradually declines
as the animal matures (Seroogy et al., 1994, supra).
[0009] Physiologically, TGF.alpha. acts on numerous cell types
throughout the body, including many of neural origin (for review,
see Derynck, 1992, supra). It supports the survival of cultured
central neurons (Morrison et al., Science 238:72-75, 1987; Zhang et
al., Cell. Regul. 1:511-521, 1990) and, unlike EGF, enhances
survival and neurite outgrowth of dorsal root ganglion sensory
neurons (Chalazonitis et al., J. Neurosci. 12:583-594, 1992). It
also stimulates proliferation and differentiation of neuronal and
glial progenitor cells from developing and adult brains (Anchan et
al., Neuron 6:923-936, 1991).
[0010] The trophic effects of EGF-family peptides on mesencephalic
dopaminergic neurons in culture have also been studied in recent
years. EGF enhances the survival of E16 dopamine neurons in mixed
midbrain cultures (Casper et al., J. Neurosci. Res. 30:372-381,
1991), but the degree to which it stimulates dopamine uptake is
modest (Knusel et al., J. Neurosci. 10:558-570, 1990). TGF.alpha.
also supports the survival of mesencephalic dopamine neurons in
dissociated cell culture, but its effect is more selective than
that of EGF (Ferrari et al., J. Neurosci. Res. 30:493-497, 1991;
Alexi and Hefti, Neurosci. 55:903-918, 1993).
[0011] Another important characteristic of EGF-family growth
factors is their ability to protect midbrain dopamine cells from
neurotoxic assaults. EGF has been shown to protect dopamine neurons
from glutamate or quisqualate excitotoxicity in dissociated cell
culture (Casper and Blum, J. Neurochem. 65:1016-1026, 1995). It has
also been demonstrated to protect cultured dopamine cells from the
selective dopamine neurotoxin, 1-methyl-4-phenylpyridinium
(MPP.sup.+; Park et al., Brain Res. 599:83-97, 1992) and to
increase dopamine uptake in MPP.sup.+-treated cultures
(Hadjiconstantinou et al., J. Neurochem. 57:479-482, 1991).
[0012] The results of studies with EGF in vivo were consistent
those obtained in culture; EGF effected neuroprotection in both
instances. For example, intracerebroventricular (ICV) infusions of
EGF reduced amphetamine-induced rotations, increased the number of
surviving tyrosine hydroxylase immunoreactive (TH-IR) cells in the
SN, and increased striatal TH-IR fibers after transection of the
nigrostriatal pathway in a rat model of PD (Pezzoli et al.,
Movement Disord. 6:281-287, 1993; Ventrella, J. Neurosurg. Sci.
37:1-8, 1993). ICV infusions of EGF into the brains of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned mice
enhanced the content of dopamine and 3,4-dihydro-zyphenylacetic
acid (DOPAC) and the activity of tyrosine hydroxylase in the
striatum (Hadjiconstantinou et al., 1991, supra; Schneider et al.,
Brain Res. 674:260-264, 1995).
[0013] Despite its more potent activity in vitro, relative to EGF,
the trophic effects of TGF.alpha. in vivo--particularly in animals,
including humans, with neurological deficits--are undetermined. The
present invention is based on newly discovered effects of
TGF.alpha. infusion on cells in the normal and abnormal (lesioned)
central nervous system, which are described herein.
SUMMARY OF THE INVENTION
[0014] The present invention features methods and compositions for
treating a patient who has a neurological deficit. The method can
be carried out, for example, by contacting (in vivo or in culture)
a neural progenitor cell of the patient's central nervous system
(CNS) with a polypeptide that binds the epidermal growth factor
(EGF) receptor and directing progeny of the proliferating
progenitor cells to migrate en masse to a region of the CNS in
which they will reside and function in a manner sufficient to
reduce the neurological deficit. The method may include a further
step in which the progeny of the neural precursor cells are
contacted with a compound that stimulates differentiation.
[0015] Other objects, advantages, and novel features of the present
invention will become apparent from the brief description of the
drawings, the detailed description of the invention, and the
working examples that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic of a coronal section of rat forebrain.
The injection pipette illustrated on the right-hand side represents
one location into which a growth factor can be infused, the
striatum (str). (cerebral cortex=ctx; lateral ventricle=lv).
[0017] FIGS. 2A-2D are photomicrographs of coronal sections through
the mesencephalon of the adult rat brain that have been probed to
show the distribution of EGF receptor mRNA. In FIG. 2A a "sense"
probe was applied as a control. In FIGS. 2B-2D, sections through
the substantia nigra (sn) reveal moderately abundant expression in
the hippocampus (hip), the medial portion of the sna, and the
parabranchial and paranigral nuclei of the ventral tegmental area
(vta). In the most-caudal midbrain (FIG. 2D), the interpeduncular
nucleus (ip) was the most intensely labeled. Scale bar=5 mm.
[0018] FIG. 3 is a photomicrograph of a coronal section through the
striatum of an adult rat brain that was infused with TGF.alpha. and
probed to show the distribution of EGF receptor mRNA. On the side
of the infusion, a dramatic increase in hybridization density is
apparent in the medial striatum adjacent to the lateral
ventricle.
[0019] FIG. 4 is a photomicrograph of a coronal section through the
forebrain of an adult rat brain that was infused with TGF.alpha.
and lesioned with 6-OHDA. In situ hybridization was performed to
localize EGF receptor mRNA, which appears as an intense ridge
extending well into the body of the striatum.
[0020] FIG. 5 is a bar graph showing the average standardized
densities of TGF.alpha. mRNA hybridization in striata in each of
five groups of animals examined (normal; aCSF infusion, no lesion;
aCSF infusion, lesion; TGF.alpha. infusion, no lesion; TGF.alpha.
infusion, lesion). The paired bars represent densities in striata
ipsilateral and contralateral to the treatment. Average
hybridization density was significantly reduced by one-quarter
ipsilateral to the treatments in both groups receiving nigral
6-OHDA lesions. The striatal infusion of TGF.alpha. peptide had no
impact on the decrease. Averages.+-.S.E.M. (Student's t-test,
paired for ipsilateral-contralateral comparisons; P values, *
p<0.005, ** p<0.001).
[0021] FIG. 6 is a bar graph showing the average standardized
densities of EGF receptor mRNA hybridization in the subependymal
regions along the edges of the striata bordering the lateral
ventricles of animals in the same five test groups described in
FIG. 5. The paired bars represent densities in striata ipsilateral
and contralateral to the treatment. Average hybridization density
was approximately doubled in the ipsilateral subependymal region in
both groups receiving TGF.alpha. striatal infusions.
Averages.+-.S.E.M. (Student's t-test, paired for
ipsilateral-contralateral comparisons; P values, * p<0.01, **
p<0.0001).
[0022] FIG. 7 is a bar graph showing the average standardized
densities of EGF receptor mRNA hybridization in the striatal
ridges, the non-ridge body of the striatum, and the subependymal
regions in all animals with striatal ridges. The paired bars
represent densities in striata ipsilateral and contralateral to the
treatment. Average hybridization density was highest in the
striatal ridge ipsilateral to the treatments. No striatal ridges
ever appeared in the contralateral striata. TGF.alpha. striatal
infusions. Averages.+-.S.E.M. (Student's t-test, paired for
ipsilateral-contralateral comparisons; P values, * p<0.005, **
p<0.001).
[0023] FIGS. 8A and 8B are photomicrographs of coronal sections
through the striatum of an adult rat that received a nigral 6-OHDA
lesion and TGF.alpha. infusion for fourteen days. In FIG. 8A,
silver staining in the caudate-putamen ipsilateral to the
treatments reveals huge numbers of cells in the dorsal portion of
the ridge, many of which exhibit an elongated morphology and are
oriented normal to the subependymal region. There is also an
increase in the number of cells along the lateral ventricle (lv).
In the contralateral striatum (FIG. 8B), the cellular population is
not expanded, either in the striatum or along the lateral
ventricle.
[0024] FIGS. 9A-9D are photomicrographs of thionin-stained coronal
sections of adult rat brain from animals that were lesioned with
6-OHDA and infused with TGF.alpha. for variable periods of time. In
FIG. 9A, after four days of infusion, cellular expansion in the
subependymal region is barely detectable above background staining.
In FIG. 9B, after six days of infusion, the aggregation of
thionin-stained cells near the lateral ventricle is much more
robust. In FIG. 9C, after nine days of infusion, a region of
densely-stained cells appears slightly lateral to the subependymal
zone at the ventral end of the cellular expansion. In FIG. 9D,
after fourteen days of infusion, a dense, well-formed ridge is
evident well into the body of the striatum.
[0025] FIGS. 10A and 10B are photomicrographs of coronal sections
of adult rat brain from animals that were lesioned with 6-OHDA and
infused with TGF.alpha. for fourteen days. In FIG. 10A, nestin
immunohistochemistry reveals an intense striatal ridge. In FIG.
10B, thionin staining of a near-adjacent section confirms the
registry between the nestin-IR cells and the striatal ridge.
[0026] FIGS. 11A-11C are photomicrographs of thionin stained
coronal sections from adult rat brain from animals that were
lesioned with 6-OHDA and infused with TGF.alpha., at varying
distances from the lateral ventricle, for fourteen days. In FIG.
11A, where the infusion cannula was implanted in the far-lateral
striatum, the ridge parallels the subependymal zone and is less
dense than that seen with a mid-striatal infusion. In FIG. 11B,
where the infusion cannula was implanted mid-striatum, the ridge is
characteristically S-shaped, with the ventral portion extending far
out into the ventral striatum. In FIG. 11C, where the infusion was
immediately adjacent to the lateral ventricle, the striatal ridge
is L-shaped and generally exhibits very dense thionin staining.
[0027] FIG. 12 is a bar graph depicting the maximum displacement of
the striatal ridge (from the lateral ventrical; in mm) in coronal
sections of adult rat brain after nigral lesion with 6-OHDA and
mid-striatal infusion of TGF.alpha. for fourteen days. Animals
treated according to "Schedule A" (left-most bar) received lesions
first, followed by TGF.alpha. infusions four to five weeks later.
Animals treated according to "Schedule B" received lesions two days
after the 14-day TGF.alpha. infusion began. Averages.+-.S.E.M.
(Student's t-test; P value, * p<0.01).
DETAILED DESCRIPTION
[0028] I. The Striatum and Nigrostriatal System
[0029] A. Anatomy, Connectivity and Neurochemistry
[0030] Within the brain, the striatum, pallidum, substantia nigra,
ventral tegmental area (VTA), subthalamic nucleus, and amygdala are
collectively referred to as the basal ganglia. The striatum
contains dorsal and ventral components, each of which is further
subdivided into additional anatomical structures. In humans, the
dorsal striatum consists of the caudate nucleus and the putamen.
The C-shaped caudate follows the curve of the lateral ventricles.
Its tail portions extend past the ends of the inferior horns and
joins the amygdala in each temporal lobe. The head of the caudate
turns ventrally from the anterior end of the anterior horns and
fuses with the putamen. Although largely anatomically distinct in
the human brain, they are combined into a common structure, the
caudate-putamen or caudoputamen, in rodents.
[0031] The ventral striatum is comprised of the nucleus accumbens,
olfactory tubercle, and the associated striatal cell bridges. The
pallidum includes the globus pallidus, entopeduncular nucleus,
substantia nigra pars reticulata (SNr), and the ventral pallidum.
The entopeduncular nucleus and SNr have very similar afferent and
efferent connections. The ventral pallidum contains regions that
have a mix of connections that are similar to both the globus
pallidus and entopeduncular nucleus. The other part of the
substantia nigra, the pars compacta (SNc), includes dopamine
neurons that span the substantia nigra-ventral tegmental area
(SN-VTA), as well as dopamine cell clusters in the SNr. The
circuitry of the basal ganglia is complex, but is very similar in
both rats and humans (Fallon and Loughlin, Cerebral Cortex, E. G.
Jones and A. Peters, Eds., Vol. 6, pp. 41-127, Plenum Press, New
York, 1987; Alheid and Heimer, Progr. Brain Res. 107:461-484,
1996), making the rat a useful model for studying connections,
neurochemistry, pharmacology, function, and clinical correlates of
this system in the mammalian brain.
[0032] The striatum, together with other nuclei of the basal
ganglia, contributes to the regulation of movement and emotion. A
number of diseases affecting the system or its innervation are
associated with profoundly debilitating motor impairment, often
accompanied by affective disorders.
[0033] The caudate and putamen are the primary input nuclei of the
basal ganglia and receive major excitatory projections from the
cerebral cortex and the centromedian and intralaminar nuclei of the
thalamus. Corticostriatal afferents are glutamatergic. Afferents
from the thalamus are also thought to be glutamatergic. The
substantia nigra pars compacta (SNc) provides dense dopaminergic
input to the striatum via the nigrostriatal pathway (for review of
this system in the rat, see Fallon and Loughlin, The Rat Nervous
System, G. Paxinos, Ed., pp. 215-237, Academic Press, San Diego,
1995). The ventral striatum and nucleus accumbens receive the bulk
of their dopaminergic innervation from dopamine cells of the VTA in
the ventromedial mesencephalon. Limbic afferents from the amygdala
and serotonergic fibers from the midbrain or raphe also terminate
in the ventral striatum.
[0034] The distribution of striatal afferents and their
terminations are not simply uniform representations of their
regions of origin. The striatum is organized into patches or
striosomes embedded in a functionally and chemically distinct
surrounding matrix. This organization was originally demonstrated
using histochemistry for acetylcholinesterase (AChE), which
selectively stains the matrix (Graybiel and Ragsdale, Proc. Natl.
Acad. Sci. USA, 75:5723-5726, 1978). Since then, enzyme
histochemistry, immunocytochemistry, in situ hybridization,
receptor binding with radiolabeled ligands, anterograde
degeneration and other methods have been used to identify many
additional markers that are differentially distributed in the two
compartments. Markers for the matrix include calbindin,
somatostatin, and dopamine uptake ([.sup.3H]mazindol binding) sites
(Gerfen, J. Comp. Neurol. 236:454-476, 1985; Voorn et al., J. Comp.
Neurol. 289:189-201, 1989). Striosomes can be identified by their
higher relative abundance of enkephalin, 5'-nucleotidase activity
with nigrostriatal lesions, tyrosine hydroxylase, mu opoid receptor
binding, and substance P (Graybiel et al., Neurosci. 6:377-397,
1981; Schoen and Graybiel, J. Comp. Neurol. 322:566-576, 1992). As
might be expected, however, there are interspecific and
developmental variations in many of these markers and some are
useful only in certain regions of the striatum.
[0035] The heterogeneity of chemical markers is further complicated
by the selective origin and termination of many striatal pathways
in the patch or matrix compartments. For instance, afferents from
motor, cingulate, somatosensory and visual areas of cortex
terminate in the matrix (Gerfen, Nature 311:461-464, 1984; Donoghue
and Herkenham, Brain Res. 365:397-403, 1986). The bulk of the
corticostriatal afferents from deep layer V and layer VI of limbic
cortex terminate in the patches while most from more superficial
layer V and layers II and III provide input to the matrix (Gerfen,
Science 246:385-388, 1989). Afferents from the VTA and the dorsal
tier of the SNc provide dopaminergic input to the matrix. The
patches receive dopamine innervation from the ventral tier of the
SNc and dopamine cell clusters in the SNr (Schoen and Graybiel, J.
Comp. Neurol. 322:566-576, 1992). In the dorsal striatum, inputs
from nuclei in the medial division of the thalamus terminate in the
patches while afferents from the lateral division--including
anterior and posterior intralaminar and rostral ventral tier
nuclei--predominantly innervate matrix tissue (Ragsdale and
Graybiel, J. Comp. Neurol. 311:134-167, 1991). In addition,
amygdalostriatal fibers originating in the basolateral nucleus of
the amygdala selectively innervate the patch compartment (Ragsdale
and Graybiel, J. Comp. Neurol. 269:506-522, 1988).
[0036] Striatal efferents are also differentially distributed with
respect to the patch-matrix organization. The striatonigral
pathway, one of the two major pathways originating in the striatum,
has been shown to be comprised of two distinct projections. Fibers
arising from neurons in the patch compartment terminate around
dopamine neurons in the ventral SNc and in dopamine cell clusters
in the SNr. Matrix neurons give rise to topographically-arranged
projections to the SNr, including non-dopaminergic areas and
dopamine neurons whose dendrites are located in the SNr (Gerfen,
1984, supra; Jiminez-Castallanos and Graybiel, Neurosci.
32:297-321, 1989).
[0037] The other major efferent projection, the striatopallidal
pathway, projects to the globus pallidus. It has not been shown to
be distributed with respect to the patch-matrix organization;
however, it is neurochemically distinct from the striatonigral
system. The majority of striatopallidal fibers express enkephalin
and not dynorphin or substance P. In contrast, few striatonigral
projections contain enkephalin, but most express dynorphin and
substance P (Gerfen and Young, Brain Res. 460:161-167, 1988). In
primates, the two systems also differ in their anatomical regions
of origin: striatopallidal efferents arise mainly from the putamen
while striatonigral efferents originate primarily in the caudate
(Parent et al., Brain Res. 303:385-390, 1984).
[0038] In addition to the heterogeneous distribution of striatal
connections, several morphologically and chemically distinct types
of neurons are found in the striatum (Albin et al., Trends
Neurosci. 12:366-375, 1989; Groves, Brain Res. Rev. 5:234-238,
1983). They are traditionally classified as either spiny or aspiny
based on their dendritic morphology. There are two generally
recognized types of spiny neurons in the striatum. They contain
various combinations of GADA, substance P, enkephalin and
dynorphin, but are predominantly GABAergic. The medium spiny
neurons (spiny type I) are by far the most abundant, comprising
90-95% of all striatal neurons. They have smooth cell bodies and
dense accumulations of spines on the distant portions of their
dendrites. Their dendritic arborizations range to about 200 .mu.m
from the somata. Medium spiny neurons are the principle terminal
targets for dopaminergic neurons in the SNc, which form synapses
predominantly on the necks of the dendritic spines. Spiny type II
neurons are much larger, with variable arbors extending up to about
600 .mu.m from the soma.
[0039] Spiny neurons are the projection neurons of the striatum.
Those in the matrix containing GABA and substance P project
predominantly to the internal segment of the globus pallidus
(GP.sub.i) and the SNr. Spiny GABAergic matrix neurons containing
enkephalin, on the other hand, innervate the external segment of
the globus pallidus (GP.sub.e). Spiny neurons in the patch
compartment send the majority of their efferents to the SNc (Albin
et al., 1989, supra).
[0040] Striatal projection neurons of the two major efferent
pathways can also be distinguished by their dopamine receptor
subtypes. Substance P/dynorphin neurons projecting to the
substantia nigra express predominantly D.sub.1 dopamine receptors,
while enkephalinergic striatopallidal neurons express mainly
D.sub.2 receptors. Neither receptor type, however, is expressed
exclusively in either projection (Besson et al., Neurosci.
26:101-119, 1989; Gerfen et al., Science 250:1429-1432, 1990).
[0041] Three recognized types of aspiny neurons make up the
population of striatal interneurons (Groves, 1983, supra;
Carpenter, In: Core Text of Neurosciences, pp. 325-360, Williams
and Wilkins, Baltimore, 1991). Together, they make up 10% or less
of the total number of neurons of the striatum. Aspiny type I
neurons are the most common of the three and have smooth dendrites
in arbors slightly smaller than those of medium spiny neurons. They
are largely GABAergic, but many contain somatostatin and
neuropeptide Y. Aspiny type II neurons are distinguished by their
large cell bodies and AChE and choline acetyltransferase (CHAT)
staining. This cell type forms symmetric synapses with medium spiny
neurons. Medium aspiny type III neurons are the least well
characterized but are thought to contain GABA. There are probably
additional chemically-defined and connectionally-defined subsets of
these classes of neurons beyond the ones already identified.
[0042] B. Topography and Development
[0043] Experiments with anterograde and retrograde tracers in
striatal projections of the mesencephalic dopamine system revealed
precise topographies in adult rodents (Fallon and Moore, J. Comp.
Neurol. 180:545-580, 1978). The dorsal striatum receives
dopaminergic innervation from neurons in the ventral and
intermediate SN and VTA. The ventral striatum and nucleus accumbens
receive dopaminergic input from the dorsal VTA and intermediate SN
(Fallon, Ann. NY Acad. Sci. 537:1-9, 1988).
[0044] Neurogenetic gradients in the developing system parallel the
topographic arrangements of projections in the mature system. The
dorsolateral portion of the SN is the earliest produced in the
embryo, before embryonic day 15 (E15) in the rat (Altman and Bayer,
J. Comp. Neurol. 198:677-716, 1981). Projections from this region
innervate the lateral and ventral regions of the striatum (Carter
and Fibiger, Neurosci. 2:569-576, 1977; Veening et al., Neurosci.
5:1253-1268, 1980), which are also the earliest striatal areas
generated (Bayer, Neurosci. 4:251-271, 1984). As the striatum is
populated with younger neurons in a ventrolateral-to-dorsomedial
gradient, afferents arrive from more ventromedial (and
later-produced) portions of the SN (generated after E15) to
innervate these later-produced striatal areas. Thus, the youngest
(ventromedial) nigral dopamine neurons innervate the youngest
(dorsomedial) striatal neurons, and older (dorsolateral) nigral
dopamine neurons innervate older (ventrolateral) striatal neurons.
This pattern is repeated in the GABAergic striatonigral projections
as well (Bunney and Aghajanian, Brain Res. 117:423-435, 1976).
[0045] The neurons of the striatum are derived from a
neuroepithelium surrounding the lateral ventricles in the prenatal
and early postnatal brain. A ventricular zone initially lines the
ventricles, later joined by the subventricular (or subependymal)
zone just deep to it. As the brain matures, the ventricular zone
disappears, but the subependymal zone persists as a thin layer of
cells. This zone contains neural stem cells and progenitor cells
that migrate along a defined and restricted path to replenish the
labile interneuronal cell population of the olfactory bulb (Luskin,
Neuron 11:173-189, 1993; Lois and Alvarez-Buylla, Science
264:1145-1148, 1994).
[0046] C. Pathology
[0047] The striatum and its dopaminergic innervation are vulnerable
to a number of conditions including several neurodegenerative
diseases (Albin et al., 1989, supra) such as Huntington's disease
(HD) and Parkinson's disease (PD). HD is an autosomal dominant
hereditary disease (chromosome 4) characterized by progressive
degeneration of the striatum. It is associated with involuntary
choreathetotic movements of the limbs and face and disruptions of
voluntary movement (for review, see Purdon et al., J. Psychiatr.
Neurosci. 16:359-367, 1994). Medium spiny GABAergic neurons in the
matrix compartment are most affected, especially the
GABA/enkephalinergic neurons projecting to the GP.sub.e (Albin et
al., 1989, supra). Large aspiny cholinergic interneurons and small
aspiny interneurons containing somatostatin, neuropeptide Y and
NADPH-diaphorase, also found in the matrix compartment, are
relatively spared (Ferrante et al., Science 230:561-563, 1985;
Reiner et al., Proc. Natl. Acad. Sci. USA 85:5733-5737, 1988). In
more advanced stages of HD, however, neuronal degeneration includes
all types of striatal neurons and extends to other nuclei of the
basal ganglia, the cerebral cortex, hypothalamus, and
cerebellum.
[0048] Parkinson's Disease (PD) is characterized by resting tremor,
rigidity, inability to initiate movement (akinesia) and slowness of
movement (bradykinesia) (Marsden, Lancet 335:948-952, 1990). The
motor deficits are associated with progressive degeneration of the
dopaminergic nigrostriatal pathway and, to various extents, loss of
dopaminergic innervation to the nucleus accumbens and degeneration
of noradrenergic cells of the locus ceruleus and serotonergic
neurons of the raphe (Javoy-Agid et al., Adv. Neurol. 40:189-198,
1984; Agid, Lancet 337:1321-1327, 1991). Up to 80% of
nigral-dopamine neurons can be lost before significant motor
deficits are manifest.
[0049] One of the major strategies for using peptides known as
neurotrophic factors as therapeutic agents in the treatment of
neurodegenerative diseases is to arrest the degenerative process
and enhance the function of remaining cells. The studies presented
in the Examples below will illustrate how the present invention
expands the use of neurotrophic factors beyond that which has been
previously suggested.
[0050] II. Treatment of Neurological Deficits
[0051] As demonstrated by the examples below, neural precursors in
the adult forebrain subependymal zone can be stimulated to
proliferate and migrate en masse into the central nervous system
(CNS) (e.g., into the striatum) in response to an infusion of a
polypeptide that binds the EGF receptor (e.g., TGF.alpha.; the term
"polypeptide" as used herein refers to any chain of amino acid
residues, regardless of length or post-translational modification).
Furthermore, one can direct migration of the proliferating cells as
a dense ridge. As described below, directed migration can be
accomplished in a variety of ways. For example, it is facilitated
by denervation of the target region (which can be achieved by
neurochemical or mechanical forces), by application of a
polypeptide growth factor (e.g., TGF.beta., which increases the
regional expression of cell adhesion molecules such as fibronectin
and laminin), and by contacting the cells along a desired migratory
path with a compound that inhibits a naturally occurring signal
that would otherwise inhibit migration (i.e., creating a permissive
microenvironment by inhibiting an inhibitor).
[0052] Moreover, the shape of the migratory ridge can be controlled
by varying the location of the infusion (e.g., by altering the
placement of the infusion cannula or of a biodegradable capsule
containing the active compound(s) of the invention). Similarly, the
number of cells within the ridge can be controlled by varying the
dosage of the active compound(s) (e.g., the dosage of TGF.alpha.)
or the distance at which it is released relative to the population
of neural precursor cells in the subependymal region. As described
below (and as illustrated in FIGS. 10A, 10B, 11A, 11B, and 11C),
when animals received mid-striatal or medial striatal infusions,
the migrating cells stopped before they reached the infusion
cannulae, which resulted in S-shaped or L-shaped ridges. Thus, it
is possible not only to facilitate the cells' migration, but to
control where that migration ends. Adjusting the dose and the
location of release of the growth factor (and perhaps other
compounds) may allow restriction of the area affected to a
relatively limited target region.
[0053] The proliferation and migration of neural precursor cells in
the adult mammalian brain are distinct events that can be
controlled separately. Intracerebroventricular (ICV) or
intrastriatal infusions of TGF.alpha. or EGF without
deafferentation can induce proliferation, but degenerating, damaged
(e.g., by deafferentation or other injury), or otherwise abnormal
(i.e., malfunctioning) cells must be present to facilitate
migration, at least on a scale that is large enough to impact
recovery from an associated neurological deficit. As described
further below, one can mimic the facilitating effect of
degenerating, damaged, or otherwise abnormal cells with
pharmacological agents. For instance, one can stimulate transient
expression of cell adhesion molecules in the striatum by
administering an inductive compound. For example, fibronectin is
strongly upregulated by transforming growth factor beta (TGF.beta.)
in cultures of cerebellar astrocytes (Baghdassarian et al., Glia
7:193-202, 1993). In transgenic mice overexpressing TGF.beta.,
fibronectin and laminin are also strongly increased in the CNS over
normal levels (Wyss-Coray et al., Am. J. Pathol. 147:53-67, 1995).
Thus, directing migration with any compound that stimulates the
expression of extracellular matrix molecules or cell adhesion
molecules, particularly along the desired path of migration, is
considered within the scope of the present invention.
[0054] Forebrain neural stem cells, which give rise to the
migrating progenitors, are believed to remain in place along the
ventricular wall (Morshead et al., Neuron 13:1071-1082, 1994). In
the experiments described below, a region of intense EGF receptor
hybridization persisted along the lateral ventricle after the
migratory ridge had moved into the striatum. In addition, elongated
cells were always found between the ridge and the lateral
ventricle. Thus, despite the mass cellular migration away from the
subependymal zone, the stem cells themselves likely were not part
of the migrating ridge. These neural stem cells would provide a
renewable source of new neurons and glia. Therefore, multiple waves
of neural progenitor cells can be stimulated to migrate into
regions of the brain that are injured, or that have degenerated or
that otherwise contribute to a neurological deficit. The
persistence of these cells also suggests that the normal function
of stem cells in the adult forebrain--presently thought to provide
new neurons for the olfactory bulbs--should not be irreversibly
disrupted by the treatments.
[0055] Abundant striatal expression of TGF.alpha. (and its mRNA)
and a lack of dopaminergic innervation also characterize the early
developing striatum (Weickert and Blum, Devel. Brain Res.
86:203-216, 1995; Bayer, Intl. J. Devel. Neurosci. 2:163-175,
1984). Similarly, the increased EGF receptor mRNA expression in the
subependymal region in TGF.alpha.-infused animals mimics the
abundant EGF receptor mRNA hybridization observed in the
periventricular neuroepithelium in the developing brain (Seroogy et
al., Neuroreport 6:105-108, 1994; Seroogy et al., Brain Res.
670:157-164, 1995). Messenger RNAs encoding forms of fibronectin,
and its receptor, and other cell adhesion molecules, which may
facilitate the migration of neural precursors, are also
developmentally regulated (Pesheva et al., J. Neurosci. Res.
20:420-430, 1988; Prieto et al., J. Cell Biol. 111:685-698, 1990;
Pagani et al., J. Cell Biol. 113:1223-1229, 1991; Linnemann et al.,
Int. J. Devel. Neurosci. 11:71-81, 1993). Thus, one way to
conceptualize the effects observed in the TGF.alpha.-infused and
6-OHDA lesioned animals in the present studies is as a selective
recapitulation of embryonic neurogenesis. That is, neural stem
cells in the adult mammalian brain may respond to proliferation
signals and their progeny may respond to migration signals as they
do in the developing animal.
[0056] Neural stem cells have recently been found in subependyma
throughout the adult rodent CNS (Weiss, Soc. Neurosci. Abstr.
25:101, 1995; Ray et al., Soc. Neurosci. Abstr. 22:394.5, 1996) and
in the subependyma of the adult human forebrain (Kirschenbaum et
al., Cerebral Cortex 4:576-589, 1994). According to the methods
described herein, these cells can be manipulated to provide a
source of new neurons for diseased, injured, or otherwise damaged
or malfunctioning CNS neurons in diverse regions of the brain and
spinal cord.
[0057] A. Advantages of the Present Invention
[0058] As described further below, one of the techniques proposed
for treating a neurological deficit involves removing neural
precursors from a patient who has such a deficit and growing those
cells in culture to generate large numbers of neural progenitors.
The cells may then be re-implanted into the same patient using
techniques known to those of ordinary skill in the art (e.g., see
Stein et al., In: Brain Repair, pp. 87-103, Oxford University
Press, New York, 1995, or Leavitt et al., Soc. Neurosci. Abstr.
22:505, 1996). Clearly, this technique is advantageous to those
presently in use that require embryonic cells from aborted fetuses;
it avoids altogether the ethical issues raised by the need to use
aborted fetuses as tissue donors. In addition, it is more likely to
succeed because it will not stimulate the immune response that is
responsible for a high incidence of transplant rejection.
[0059] Stimulating proliferation and migration of neural precursors
in vivo has additional advantages; in vivo stimulation reduces the
extent and possibly the number of invasive neurosurgical
procedures. No stem cell excision surgery would be performed and
multiple plugs of transplanted cells, which are typically required
with embryonic or cultured cell grafts, would not be necessary.
Further, there would be no massive die-off of undifferentiated
neural progenitor cells due to the transplantation procedure.
Typically, with human fetal dopaminergic cell grafts, 90% to over
99% of the implanted cells die before they become established in
the host brain (Freed et al., Soc. Neurosci. Abstr. 22:481.3,
1996).
[0060] Another advantage provided by the present invention is that
neural progenitor cells would not be isolated from the host brain
by scar tissue. Plugs of transplanted cells become encapsulated
within an envelope of glyotic scar tissue and reactive astrocytes.
In addition to the physical barrier of the dense glyotic tissue,
reactive astocytes within the scar tissue release factors which
inhibit neurite outgrowth (McKeon et al., 1995). Neural progenitors
created in vivo are not isolated from the rest of the brain by scar
tissue. The outgrowth of their neurites, therefore, would not be
inhibited by a massive proliferation of reactive astrocytes. The
directed migration provided for herein therefore allows selective
repopulation (which may vary in extent) of specific injured regions
of the CNS with large numbers of new cells without disturbing
undamaged areas.
[0061] The techniques presented here also represent an advance over
the single previous study of forebrain neural stem cells stimulated
in vivo. In that study, adult rats received ICV infusions of EGF
for six days and were followed for up to seven weeks post-infusion
(Craig et al., J. Neurosci. 16:2649-2658, 1996). In the present
study, TGF.alpha. was infused for fourteen days and followed for up
to three months following the end of infusion. This difference is
critical in that only in the present study did the cells of the
periventricular expansion migrate en masse into the overlying
striatum. The directed mass migration of neural progenitors into a
selected target area represents a much preferred method to
repopulate degenerated brain regions with new neurons.
[0062] One area of intense recent interest is the manipulation of
neural stem cell differentiation. Both the final location and the
neurochemical phenotypes of the cells once they have differentiated
are of primary importance and are discussed further below.
[0063] When neural precursor cells were removed from adult rodent
brains and differentiated in vitro, cells immunochemically
identified as astrocytes, oligodendrocytes and neurons are seen
(Reynolds and Weiss, Science 255:1707-1710, 1992; Reynolds et al.,
J. Neurosci. 12:4565-4574, 1992; Lois and Alvarez-Buylla, Proc.
Natl. Acad. Sci. USA 90:2074-2077, 1993). Many of the cells
identified as neurons were also immunoreactive for GABA and
substance P, neurochemical markers for two cell types normally
found in the striatum. Precursor cells explanted from the adult
human brain also expressed neuronal markers and displayed
electrophysiological properties associated with neurons
(Kirschenbaum et al., Cerebral Cortex 4:576-589, 1994).
[0064] These experiments suggest that when cells of the striatal
ridge spontaneously differentiate in vivo, many of them will become
cells with phenotypes typical of striatal neurons. Some recent data
suggests that their phenotypes can be altered by exposure to
different combinations of neurotrophins (Lachyankar et al., Soc.
Neurosci. Abstr. 22:394.7, 1996). Progenitor cells receiving
different treatments expressed different neurochemical
immunomarkers once they differentiated, including
acetylcholinesterase, GABA, tyrosine hydroxylase (TH), and
calbindin. The expression of TH was particularly interesting, since
combined proliferation, migration and directed differentiation into
dopamine cells could provide a novel method to replace striatal
dopamine lost in Parkinson's disease (PD).
[0065] In PD patients, functionally significant numbers of new
dopamine-producing striatal cells would aid in the reversal of
motor deficits in a manner similar to transplants of aborted fetal
midbrain tissue. In patients with Huntington's disease (HD), neural
precursors would be stimulated to repopulate the striatum with new
medium spiny GABAergic and other neurons lost to the disease. Some
recent evidence from a different line of research indicates that
reconstruction of the striatopallidal pathway itself might be
possible. Conditionally immortalized neural progenitor cells
transplanted into the striatum differentiated and sent processes
from the striatum to the globus pallidus (Lundberg et al.,
1996).
[0066] B. Neurological Deficits Amenable to Treatment
[0067] Because the invention rests on the discovery that
multipotent precursor cells can be stimulated to divide and migrate
through the brain, it can be used to treat neurological deficits
caused by a wide variety of diseases, disorders, and injuries.
These insults include, but are not limited to, the following
(others of skill in the art may categorize differently the diseases
and disorders listed below; however categorized, the neurological
deficits with which they are associated are amenable to treatment
according to the methods of the present invention).
1. Degenerative Diseases
[0068] Degenerative diseases that can be treated according to the
methods of the invention include Alzheimer's disease (AD),
Parkinson's disease (PD), Huntington's disease (HD), Pick's
disease, progressive supranuclear palsy (PSP), striatonigral
degeneration, cortico-basal degeneration, childhood disintegrative
disorder, olivopontocerebellar atrophy (OPCA; including a heritable
form), Leigh's disease, infantile necrotizing encephalomyelopathy,
Hunter's disease, mucopolysaccharidosis, various leukodystrophies
(such as Krabbe's disease, Pelizaeus-Merzbacher disease, and the
like), amaurotic (familial) idiocy, Kuf's disease, Spielmayer-Vogt
disease, Tay Sachs disease, Batten disease, Jansky-Bielschowsky
disease, Reye's disease, cerebral ataxia, chronic alcoholism,
beriberi, Hallervorden-Spatz syndrome, cerebellar degeneration, and
the like.
2. Traumatic and Neurotoxic Injuries to the Central Nervous
System
[0069] Traumatic and neurotoxic injuries that can be treated
according to the methods of the invention include gunshot wounds,
injuries caused by blunt force, injuries caused by penetration
injuries (e.g., stab wounds), injuries caused in the course of a
surgical procedure (e.g., to remove a tumor or abscess from the CNS
or to treat epilepsy), poisoning (e.g., with MPTP or carbon
monoxide), shaken-baby syndrome, adverse reactions to medication
(including idiosyncratic reactions), drug overdose (e.g., from
amphetamines), post-traumatic encephalopathy, and the like.
3. Ischemia
[0070] Any disruption of blood flow or oxygen delivery to the
nervous system can injure or kill cells, including neurons and
glial cells, therein. These injuries can be treated according to
the methods of the present invention and include injuries caused by
a stroke (including a global stroke (as may result from cardiac
arrest, arrhythmia, or myocardial infarction) or a focal stroke (as
may result from a thrombus, embolus, hemorrhage, or other arterial
blockage)), anoxia, hypoxia, partial drowning, myoclonus, severe
smoke inhalation, dystonias (including heritable dystonias),
acquired hydrocephalus, and the like.
4. Developmental Disorders
[0071] Developmental disorders that can be treated according to the
methods of the invention include schizophrenia, certain forms of
severe mental retardation, cerebral palsy (whether caused by
infection, anoxia, premature birth, blood type incompatibility:
etc. and whether manifest as blindness, deafness, retardation,
motor skill deficit, etc.), congenital hydrocephalus, metabolic
disorders affecting the CNS, severe autism, Down Syndrome,
LHRH/hypothalamic disorder, spina bifida, and the like.
5. Disorders Affecting Vision
[0072] Disorders affecting vision, particularly those caused by the
loss or failure of retinal cells, can be treated according to the
methods of the invention. These disorders include diabetic
retinopathy, serious retinal detachment, retinal damage associated
with glaucoma, traumatic injury to the retina, retinal vascular
occlusion, macular degeneration, heritable retinal dystrophies,
optic nerve atrophy, and other retinal degenerative diseases.
6. Injuries and Diseases of the Spinal Cord
[0073] Injuries to or diseases affecting the spinal cord can also
be treated according to the methods of the invention. Such injuries
or diseases include post-polio syndrome, amyotrophic lateral
sclerosis, nonspecified spinal degeneration, traumatic injury (such
as those caused by automobile or sporting accidents), including any
injury that crushes, partially severs, completely severs, or
otherwise adversely affects the function of cells in the spinal
cord), injuries caused by surgery to the spinal cord (e.g., to
remove a tumor), anterior horn cell disease, paralytic diseases,
and the like.
7. Demyelinating or Autoimmune Disorders
[0074] Neurological deficits caused by demyelination or an
autoimmune response can be treated according to the methods of the
invention. Such deficits can be caused by multiple sclerosis,
possibly lupus, and others.
8. Infectious or Inflammatory Diseases
[0075] Neurological deficits caused by an infection or inflammatory
disease can be treated according to the methods of the invention.
Infections or inflammatory diseases that can cause treatable
deficits include Creutzfeldt-Jacob disease and other slow virus
infectious diseases, AIDS encephalopathy, post-encephalitic
Parkinsonism, viral encephalitis, bacterial meningitis and
meningitis caused by other organisms, phlebitis and
thrombophlebitis of intracranial venous sinuses, syphilitic
Parkinsonism, tuberculosis of the CNS, and the like.
9. Miscellaneous
[0076] Those of ordinary skill in the art are well able to
recognize neurological deficits, regardless of their cause, and to
apply the methods of the present invention to treat patients who
have such deficits. In addition to the conditions listed above,
which are amenable to treatment with the methods described herein,
neurological deficits can be caused by Lesch-Nyhan syndrome,
myasthenia gravis, various dementias, numerous parasitic diseases,
epilepsy, and the like. The methods of the invention can be readily
applied to alleviate neurological deficits caused by these and
other diseases, disorders, or injuries.
[0077] C. Polypeptides That Bind the EGF Receptor
1. The EGF Family
[0078] Polypeptides in the EGF family appear, in some ways,
unrelated. For example, TGF.alpha. and EGF have only 30% structural
homology (Marquardt et al., Science 223:1079-1082, 1984). However,
they display similar binding kinetics for, and stimulate
tyrosine-specific phosphorylation of, the M.sub.r 180,000 EGF
membrane receptor (Cohen et al., J. Biol. Chem. 255:4834-4842,
1980; Reynolds et al., Nature 292:259-262, 1981). The functional
equivalence of the two growth factors is partly attributed to the
same relative positioning of six cysteine residues, represented by
"C" in the concensus sequence:
CX.sub.7CX.sub.4,5CX.sub.10CXCX.sub.8C. These conserved residues
impose similar disulfide bond-mediated structural constraints and,
thus, a related three-dimensional structure (Twardzik et al., Proc.
Natl. Acad. Sci. USA 82:5300-5304, 1985). Those of ordinary skill
in the art are well able to compare any given amino acid sequence
with the EGF-family concensus sequence to determine whether a
polypeptide is likely to be functionally equivalent to EGF (and, if
so, useful in practicing the methods of the present invention).
(see, e.g., Blomquist et al., Proc. Natl. Acad. Sci. USA
81:7363-7367, 1984, for a description of a computer search that
revealed a similar pattern of cysteine and glycine residues in EGF,
TGF.alpha., and the sequence of a 19 kDa early protein of vaccinia
virus).
[0079] In addition to EGF, TGF.alpha., and vaccinia growth factor
(VGF), the EGF family is known to include amphiregulin (AR),
betacellulin (BTC), epiregulin (ER), heparin-binding EGF-like
growth factor (HB-EGF), schwannoma-derived growth factor (SDGF),
HUS 19878, myxomavirus growth factor Shope fibroma virus growth
factor, and teratocarcinoma-derived growth factor-1 (TDGF-1; also
known as Cripto-1 (CR-1).
2. Methods for Determining EGF Receptor Binding
[0080] Those of ordinary skill in the art are readily able to
determine whether any given polypeptide binds the EGF receptor. As
used herein, the term "binds" refers to any specific interaction
between a polypeptide and the EGF receptor that results in signal
transduction sufficient to elicit a biological response, preferably
a response that contributes to the reduction of a neurological
deficit. Preferably, any given polypeptide useful in the methods of
the present invention will bind the EGF receptor with an affinity
that is equivalent to at least 50%, more preferably at least 70%,
and most preferably at least 90% of the binding affinity of EGF
itself (see Twardzik et al., supra, for a comparison of the
biological activity of VGF, TGF.alpha., and EGF in EGF receptor
binding).
[0081] If guidance is required in performing an EGF receptor
binding assay, those of skill in the art can consult any one of
numerous publications describing a suitable procedure (the five
publications on this topic that follow are hereby incorporated by
reference in their entirety). For example, one could consult Cohen
and Carpenter, Proc. Natl. Acad. Sci. USA 72:1317-1321, 1975) or,
for a modification thereof, Twardzik et al., supra. Similarly, for
review of the EGF receptor, including specific binding and sequence
information, signalling, and receptor topology, one may consult,
for example, McInnes and Sykes (Biopolymers 43:339-366, 1997)
Boonstra et al., (Cell Biol. Intl. 19:413-430, 1995), or Gill (Mol.
Reprod. Dev. 27:46-53, 1990).
[0082] D. Directed Migration
[0083] The examples below also provide evidence for successful
directed migration of neural precursor cells, particularly in the
adult rodent forebrain. The immunohistochemical and other
techniques employed in the working examples below (and described at
length therein), as well as comparable techniques routinely
performed by those of ordinary skill in the art, can be used to
characterize the effect of any infusion of growth factor or any
other stimulus applied to direct cellular migration. Indeed, it is
possible to trace the cells' migration in some detail (i.e., the
number of cells, their size, shape, and position within the nervous
system can be determined).
[0084] A variety of stimuli can be,applied to cells in vivo to
direct their migration en masse (the term "en masse," when used
herein to describe cellular migration, refers to the movement of a
population of cells in substantially the same direction for a
sufficient period of time to be visualized as a mass (as, e.g., is
apparent in FIGS. 9, 10, and 11)). Broadly, one can direct
migration in one of two ways: (1) in a conducive manner, i.e., by
applying a stimulus that positively attracts migrating cells (such
as a chemoattractant, neurotropic factor, or a compound (e.g.,
TGF.beta.) that increases the expression of a cell adhesion
molecule or extracellular matrix molecule (e.g., fibronectin,
laminin, or a neural cell adhesion molecule)), or (2) in a
permissive manner, i.e., by applying a stimulus that inhibits a
signal that would otherwise inhibit migrating cells.
[0085] The stimuli that direct migration include disruption of the
tissue in the target area (which may be the site where cells have
been damaged, e.g., the striatum or substantia nigra, where
dopaminergic cells are known to be lost in association with a
number of debilitating neurodegenerative diseases; the cerebral
cortex, where neurons and glia are lost following an ischemic
episode caused by, e.g., a thrombus or embolus; or the spinal cord,
where motor neurons are lost due to, e.g., a traumatic injury; or
may be any site where cells make abnormal connections due to a
developmental disorder). Alternatively, tissue may be disrupted in
or along a path extending from the source of the neural progenitor
cells to the desired endpoint of their migration.
[0086] The tissue may be disrupted by physical force (e.g.,
ablating or excising neurons, or severing one or more of the
processes that extend from the neuronal cell bodies) or by applying
a chemical substance such as a toxin or neurotoxin (e.g., ricin or
6-OHDA), a corrosive chemical (e.g., an acidic or basic solution),
a compound that induces apoptosis (see, e.g., Leavitt et al., Soc.
Neurosci. Abstr. 22:505, 1996),.a compound that induces
demyelination (see, e.g., Lachapelle et al., Soc. Neurosci. Abstr.
23:1689, 1997), or a compound capable of inhibiting the activity of
the cell, e.g., an antisense oligonucleotide (such as an
oligonucleotide that inhibits transcription of the gene encoding
the cell's primary neurotransmitter), an antibody, or a
polypeptide. Many such compounds are known to those of ordinary
skill in the art and include compounds that bind to, but fail to
activate, a receptor on the cell surface, such as the metabotropic
receptors normally bound by glutamate. For example, in the studies
described below, the effect of dopamine denervation with 6-OHDA
(together with infusion of TGF.alpha.) on cellular migration is
apparent.
[0087] Those of ordinary skill in the art are well able to direct
cellular migration by applying any of the chemical substances
described above to a targeted area of the nervous system,
particularly given the remarkably clear and accurate images of a
patient's brain and spinal cord that can now be generated with,
e.g., magnetic resonance imaging or computed tomographic scans.
[0088] Moreover, it is apparent from the studies described below
that altering one or more of the variables associated with
application of a compound that directs migration (those variables
including the nature, position, concentration, and duration of the
application) can be altered to direct more precisely the migratory
path the cells follow and to define the place at which they come to
rest.
[0089] E. Differentiation Factors
[0090] While some neural precursor cells may spontaneously
differentiate, given enough time, substantially greater benefit can
be realized by controlling when and where differentiation takes
place; exerting such control allows one to limit neural
"repopulation" (which may be partial or complete, so long as it is
sufficient to reduce a neurological deficit) to areas of the CNS in
need thereof. Accordingly, various stimuli can be administered
before, during, or after contacting neural progenitor cells with a
polypeptide that binds the EGF receptor.
[0091] Broadly, the stimuli inducing differentiation can be
"general" or "directed." A general differentiation stimulus is one
that stimulates a cell to differentiate as it naturally would and
is applied whenever a neurological deficit can be reduced by
stimulating a cell to express its natural phenotype. For example,
it is known in the art that cells from the striatal subependymal
zone differentiate into GABAergic neurons upon exposure to general
differentiation signals. Thus, stimulating these cells to
differentiate by applying a general differentiation factor would
reduce the neurological deficits associated with Huntington's
Disease; in these patients, GABAergic medium spiny neurons are lost
selectively.
[0092] Those of ordinary skill in the art are well able to apply
the known means of stimulating general differentiation to stimulate
differentiation of the proliferating and migrating cells of the
present invention. For example, contacting cells with a
retinoblastoma protein is known to cause them to exit the cell
cycle, a requirement for differentiation (for a recent study, see,
Slack et al., J. Cell Biol. 140:1497-1509, 1998) and contacting
cells with a cell cycle associated kinase inhibitor, p21, can
maintain cells in a post-mitotic (i.e., differentiated) state
(Berger et al., Soc. Neurosci. Abstr. 22:505, 1996). Another
stimulus that can be applied to stimulate differentiation in the
context of the present methods is a cyclin D cell cycle regulator
(Ouaghi et al., Soc. Neurosci. Abstr. 22:1706, 1996). Should one
wish to stimulate differentiation of oligodendrocyte precursors,
integrins may be applied (see, e.g., Buttery et al., Soc. Neurosci.
Abstr. 22:1723, 1996). Brain-derived neurotrophic factor (BDNF) and
retinoic acid (RA) are well known for their abilities to stimulate
cellular differentiation (see, e.g., Ahmed et al., J. Neurosci.
15:5765-5778, 1995).
[0093] In the event neural precursor cells are cultured prior to
directing migration, they may be transplanted into an area of the
brain that is capable of influencing their differentiation. For
example, a higher percentage of transplanted neural precursor cells
differentiated into neurons when placed near the subventricular
zone (up to 35%) compared to those transplanted to more lateral
sites (where only 0-8% of the cells differentiated) (Catapano and
Macklis, Soc. Neurosci. Abstr. 23:345, 1997). Similarly,
transplanted cerebellar precursors express markers for hippocampal
neurons when they are transplanted into the hippocampus
(Vicario-Abejon et al., J. Neurosci. 15:6351-6363, 1995).
Accordingly, one of ordinary skill in the art will appreciate that,
as an alternative to contacting the progeny of proliferating neural
progenitor cells with a compound that stimulates their
differentiation, the invention can be practiced by transplanting
cells in or sufficiently near a region of the brain that is capable
of directing their differentiation.
[0094] A directed differentiation stimulus is one that stimulates a
cell to differentiate, but with a phenotype that is different from
the one it would naturally express. A directed differentiation
factor would be applied whenever a neurological deficit could be
reduced by stimulating a cell to express a non-natural phenotype.
One such instance is in the case of Parkinson's disease, where
differentiation of striatal cells into dopamine-producing cells
could substitute for the loss of dopaminergic innervation from the
substantia nigra. Similarly, one would aim to stimulate expression
of a cholinergic phenotype in the septal region, where cells are
selectively lost in Alzheimer's Disease; in spinal motor neurons,
which are lost in amyotropic lateral sclerosis and following
traumatic spinal injuries; and in oligodendrocytes, which are lost
in demyelination disorders such as multiple sclerosis.
[0095] Factors from the GDNF/neurturin (TGF.beta.) family, which
are derived from a glial cell line, may induce differentiation in
neural cells (which is, moreover, enhanced by RA) (Hishiki et al.,
Cancer Res. 58:2158-2165), and GDNF stimulates motor neuron
differentiation in rat ventral mesencephalic cultures. BDNF and
ciliary neurotrophic factor (CNTF) also promote motor neuron
differentiation (their effects appear to be additive or synergistic
to the effects of GDNF) (Zurn et al., J. Neurosci. Res. 44:133-141,
1996). In addition, motor neuron differentiation can be induced by
application of vitronectin, which is expressed in the ventral
region of the neural tube (Martinez-Morales et al., Development
124:5139-5147) and by the protein encoded by sonic hedgehog (Tanabe
et al., Curr. Biol. 5:651-658, 1995). A member of the sonic
hedgehog family, Indian hedgehog, is expressed in developing and
mature retina and promotes retinal progenitor proliferation and
photoreceptor development (Levine et al., J. Neurosci.
17:6277-6288, 1997).
[0096] Cortical neural progenitors adopt a region-specific
phenotype influenced by EGF, TGF.alpha. and the type of substrate
upon which they are grown. EGF or TGF.alpha. doubled the percentage
of limbic neurons derived from non-limbic-area precursors when they
were plated on growth factor-deficient Matrigel.TM. or collagen
type IV (Ferri et al., Development 121:1151-1160, 1995).
[0097] Insulin is known to affect differentiation of fetal neuron
cell cultures, even more than IGF-1 (Abboud et al., Soc. Neurosci.
Abstr. 23:1425, 1997). Basic fibroblast growth factor (bFGF) and
neurotrophins can be used to direct the differentiation of
hippocampal cells (Vicario-Abejon et al., Neuron 15:105-114,
1995).
[0098] In one embodiment, the methods of the invention can be
applied to restore neural pathways that are lost to degenerative
illness. For example, differentiated striatal GABAergic neurons can
restore striatopallidal projections upon their differentiation.
Those of ordinary skill in the art are well able to recognize
numerous neural pathways that are amenable to reconstruction by the
methods of the present invention.
[0099] F. Pharmaceutical Compositions
[0100] Polypeptides suitable for use in the present invention
(i.e., those that bind the EGF receptor or stimulate
differentiation, also referred to herein as "active compounds") can
be incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise one or more
polypeptides and a "pharmaceutically acceptable carrier," a term
which is intended to include any and all solvents, dispersion
media, coatings, antibacterial agents, antifungal agents, isotonic
and absorption delaying agents, and the like, that are compatible
with pharmaceutical administration. The use of such media and
agents is well known in the art. Except insofar as any conventional
media or agent is incompatible with the active compound, use
thereof in the compositions is contemplated.
[0101] Those of ordinary skill in the art appreciate the need to
formulate pharmaceutical compositions for their intended route of
administration (which may include parenteral, e:g., intravenous,
intradermal, or intramuscular injection; oral administration; or
direct application to the affected area). It is contemplated that
the present methods will be carried out by applying polypeptides to
neural precursors harvested from the brain and placed in culture or
directly to the precursor cells in vivo (by, e.g., infusion through
an injection cannula or shunt, or by implantation within a carrier,
e.g., a biodegradable capsule) but other routes of administration,
particularly parenteral (preferably intravenous) administration,
are also within the scope of the invention.
[0102] Solutions or suspensions useful in the pharmaceutical
compositions of the present invention (e.g., in a composition
containing a polypeptide that binds the EGF receptor and a compound
that stimulates the differentiation of neural precursors) can
include: sterile diluents such as water, normal saline, fixed oils,
polyethylene glycols, glycerine, propylene glycol, or other
synthetic solvents; antibacterial or antifungal agents such as
benzyl alcohol, parabens (e.g., methyl parabens), chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like; antioxidants such
as ascorbic acid or sodium bisulfite; chelating agents such as
EDTA; buffers such as acetates, citrates, or phosphates; and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide.
[0103] Pharmaceutical compositions suitable for injection include
sterile aqueous solutions (where the active compound is water
soluble) or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions. For
intravenous administration, suitable carriers include physiological
saline, bacteriostatic water, Cremophor EL.TM. (BASF; Parsippany,
N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be sterile and should be fluid to the extent that
easy syringability exists (proper fluidity can be maintained, for
example, by using coatings such as lecithin, by maintaining a
certain particle size in the case of dispersion, and by including
surfactants). The composition must be stable under the conditions
of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi,
as described above. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
[0104] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a polypeptide that binds
the EGF receptor) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying, which yields a powder of the active compound
plus any additional desired ingredient from a previously
sterile-filtered solution.
[0105] In one embodiment, the active compound is prepared with one
or more carriers that will protect it against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparing such formulations will be
apparent to those skilled in the art. The materials can also be
obtained commercially, for example, from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0106] It is especially advantageous to formulate the compositions
of the invention in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound, the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for the treatment of individuals.
[0107] In lieu of direct application of polypeptides that bind the
EGF receptor or stimulate cellular differentiation, nucleic acid
molecules encoding those polypeptides can be inserted into vectors
and used as gene therapy vectors. Gene therapy vectors can be
delivered to a subject by, for example, intravenous injection,
local administration (U.S. Pat. No. 5,328,470) or by stereotactic
injection (see, e.g., Chen et al., Proc. Natl. Acad. Sci. USA
91:3054-3057, 1394). The pharmaceutical preparation of the gene
therapy vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded, for example, in the brain or spinal
cord. Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system.
[0108] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0109] G. Treatment Regimes
[0110] By way of example (those of ordinary skill in the art are
well able to extrapolate from one model (be it an in vitro or in
vivo model) to another, progressing toward optimal dosages for
human patients), for a rodent brain, the infusions to stimulate
proliferation of neural precursor cells were continued for a period
of at least two weeks. The result being a dramatic increase in the
numbers of undifferentiated progenitor cells along the adjacent
ventricle. The continuous TGF.alpha. infusion applied in the
working examples below also supports radial cellular migration, but
is not sufficient, by itself, to stimulate the massive radial
migration observed in certain animals. The duration of any
treatment performed according to the methods described herein can
be varied according to the desired results. For example, in the
working examples below, the cells greatly increased their numbers
for more than a week prior to their mass migration away from the
ventricle. Moreover, delayed migration was facilitated by
denervation of the target region, either neurochemically or
mechanically, and may also be facilitated pharmacologically by the
concurrent infusion of factor(s) such as another neurotrophic
factor, TGF.beta., which increases the regional expression of cell
adhesion molecules believed to underlie the radial migration (as
described above). The pattern of migration and the final location
of the ridge of migrating cells can be controlled by altering the
location of the infusion.
[0111] Those of ordinary skill in the art are well able to
determine the required dosage of a compound administered in the
context of the present invention. Preferably, the dosage will
range, whether infused or released from a time-release vesicle,
from 1 to 100 ng/kg/day of the active compound or ingredient (e.g.,
TGF.alpha. or any of the factors described above); more preferably,
1 to 50 ng/kg/day; and most preferably, 1 to 10 ng/kg/day will be
administered.
EXAMPLES
Example 1
Expression of TGF.alpha. and EGF Receptor mRNAS in the Normal
Developing and Adult Nigrostriatal System
[0112] As reviewed in the Detailed Description, mRNAs encoding
EGF-family neurotrophic factors are developmentally regulated in
the nigrostriatal system. In the studies described below, the
expression of TGF.alpha. and EGF receptor mRNAs is examined in the
normal developing and adult rodent system.
[0113] A. Animals and Tissue Preparation
[0114] Adult male adult and timed-pregnant female Sprague-Dawley
rats (250-350 g) were obtained from Simonsen (Gilroy, Calif.). For
these experiments and all others described herein, the animals were
maintained in a temperature and humidity controlled vivarium. Use
of the animals for all of the experimental procedures employed was
approved by the University of California, Irvine, Animal Research
Committee in accordance with National Institutes of Health
guidelines.
[0115] Newborn (P0), postnatal day 1 (P1), and P4 animals were
anesthetized by hypothermia and sacrificed by decapitation. P10,
P21, and adult animals were sacrificed by decapitation. Their
brains were quickly removed, frozen in isopentane at -20.degree.
C., and stored at -70.degree. C. Coronal cryostat sections were cut
at 20 .mu.m and thaw-adhered to Vectabond.TM. (Vector Labs, Inc.)
coated slides in ordered anterior-to-posterior rows. The sections
were postfixed with 4% paraformaldehyde in 0.1 M phosphate buffer
(pH 7.4) for 1 hour, rinsed in phosphate buffer and air dried.
Sections were stored with desiccant at -20.degree. C until
processed.
[0116] B. Hybridization Probes
[0117] TGF.alpha. mRNA probes were generated from a 550 nucleotide
XbaI/BamHI cDNA fragment from the 5' end of rat TGF.alpha. and
subcloned into pGEM 7Zf (Promega, Inc.). Antisense and sense probes
were transcribed with SP6 and T7 polymerases, respectively. Rat EGF
receptor mRNA probes were produced from a 718 base pair BamHI/SphI
insert from the 5' end of the gene, in pGEM 7Zf. Probes for rat TH
were created using the 1.2 kb BamHI/EcoRI fragment subcloned into
pGEM 7Zf. Antisense subclones for EGF receptor and TH were
transcribed with T7 polymerase. Sense subclones for EGF receptor
and TH were transcribed with SP6 polymerase. All probes were
radiolabeled by transcription in the presence of [.sup.35S]UTP (NEN
Research Products, Inc.).
[0118] C. In situ Hybridization and Analysis
[0119] In situ nucleic acid hybridization was performed according
to the method described by Simmons et al., (J. Histotech.
12:1169-181, 1989) except that developing brains were treated with
0.0001% proteinase K solution and 0.05 M EDTA. Sections were
hybridized overnight at 65.degree. C. with sense or antisense
probes at a concentration of 10.sup.7 cpm/ml. Adjacent sections
from the same animals were hybridized to each of the probes so that
direct comparisons could be made of their anatomical
distributions.
[0120] Slides from developing and adult animals were grouped
together and apposed with .sup.14C-labeled brain paste standards to
autoradiographic BetaMax Hyperfilm (Amersham, Inc.) for six to
seven days. After successful development of the autoradiography
film, the slides were dipped in Kodak NTB-2 emulsion and exposed
for four weeks. The autoradiographic sheet film and NTB-2 emulsion
were developed with D-19 developer and Rapid Fix (Kodak, Inc.). The
brain sections were then counterstained with thionin and
coverslipped. Dipped and stained sections were examined
semiquantitatively and photographed under bright and dark field
microscopy.
[0121] Expression of TGF.alpha. and EGF receptor mRNAs in the
nigrostriatal system was traced through selected time points from
early postnatal development to adulthood. TGF.alpha. mRNA
hybridization was found in abundance in the early postnatal
striatum but was gradually reduced to near adult levels by P21.
Expression in the corpus callosum increased through postnatal
development to levels comparable to those in the striatum.
TGF.alpha. mRNA was not detected in significant abundance in the
developing or the adult substantia nigra.
[0122] Striatal EGF receptor mRNA peaked early in postnatal
development and decreased again by P21. EGF receptor was highest in
the neuroepithelia around the lateral ventricles, but was also
found at moderate levels in the body of the striatum. In the
developing ventral midbrain, EGF receptor mRNA was barely
detectable in early postnatal brains, but gradually increased to
moderate levels by P21.
[0123] In adult animals, TGF.alpha. mRNA expression was moderate in
the striatum and low-to-moderate in the ventral striatum and
nucleus accumbens. EGF receptor mRNA hybridization was found at low
levels in the body of the striatum and nucleus accumbens with
higher punctate expression dispersed throughout. It persisted at
moderate levels in the regions of striatum immediately bordering
the lateral ventricles.
[0124] In the adult ventral midbrain, EGF receptor mRNA
hybridization was found in the substantia nigra (SN), particularly
the medial pars compacta, and the paranigral and parabranchial
nuclei of the ventral tegmental area (VTA).
[0125] Previous studies indicated that TGF.alpha. and EGF receptor
mRNAs are strongly regulated during ontogeny of the nigrostriatal
system and that their expression in the adult largely represents a
continuation of the developmental pattern (Lazar and Blum, J.
Neurosci. 12:1688-1697, 1992; Weickert and Blum, Devel. Brain Res.
86:203-216, 1995). TGF.alpha. and EGF receptor mRNA hybridization
in the developing and adult animals closely paralleled the findings
of these earlier reports. The persistence of their expression in
the adult striatum and midbrain is consistent with a supportive
role in the mature nigrostriatal system.
[0126] The moderate EGF receptor mRNA expression in the adult
subependymal regions along the forebrain lateral ventricles
suggests a role in the maintenance or function of cells in this
region as well (Seroogy et al., Brain Res. 670:157-164, 1995;
Weickert and Blum, 1995, supra). TGF.alpha. (or EGF) has been shown
to support the survival and differentiation of "EGF-responsive"
cells from this region when they are explanted and grown in vitro
(Reynolds and Weiss, Science 255:1707-1710, 1992; Reynolds et al.,
J. Neurosci. 12:4565-4574, 1992). It may perform a similar function
in vivo during development.
Example 2
Modulation of TGF.alpha. and EGF Receptor mRNA Expression by
6-hydroxydopamine Lesion and Striatal TGF.alpha. Infusion
[0127] In situ hybridization was used to determine whether
nigrostriatal TGF.alpha. or EGF receptor mRNA was altered by
intrastriatal infusion of TGF.alpha.. In addition, the influence of
unilateral 6-OHDA lesions on receptor expression in infused and
uninfused animals was examined.
[0128] A. Treatment Groups
[0129] Adult male Sprague-Dawley rats weighing 250-300 grams were
obtained from Simonsen (Gilroy, Calif.) and assigned to one of five
treatment groups: (1) striatal TGF.alpha. infusion, nigral 6-OHDA
lesion (hereafter, "lesion"); (2) TGF.alpha. infusion, no lesion;
(3) artificial cerebrospinal (aCSF) infusion, lesion; (4) aCSF
infusion, no lesion; (5) no infusion, no lesion. Four to eight
animals were used per experimental group. The animals were
monitored after each surgical procedure until fully recovered and
maintained at all other times in a temperature and humidity
controlled vivarium.
[0130] B. 6-hydroxydopamine Lesions
[0131] Rats were anesthetized with 8 mg xylazine and 100 mg
ketamine per kilogram body weight. A chilled solution of 4.8 mg/ml
6-hydroxydopamine HCl (6-OHDA; Sigma Chemical Co.) in 0.9% saline
with 0.01% ascorbic acid was prepared immediately before injection.
Using sterile technique, an 8 .mu.l volume was stereotaxically
infected into the left substantia nigra (+3.7 A/P; +2.1 M/L; +2.0
D/V) at a rate of 1 .mu.l/minute using interaural zero as a
reference (Paxinos and Watson, The Rat Brain in Stereotaxic
Coordinates, Academic Press, San Diego, 1986). The success and
extent of 6-OHDA lesions were monitored by tyrosine hydroxylase
(TH) mRNA in situ hybridization in the midbrain. TH is the
rate-limiting enzyme in the dopamine synthetic pathway and is a
common marker for dopamine-producing neurons. One animal with an
incomplete lesion (retaining significant numbers of nigral TH-IR
cells) was excluded from the study and is not included in the total
number of animals.
[0132] C. Infusions
[0133] Osmotic minipumps (model 2002, Alzet, Inc.) were implanted
four to five weeks post-lesion. The minipumps were filled with
approximately 200 .mu.l of either 0.05 .mu.g/ml TGF.alpha. in
artificial cerebrospinal fluid (aCSF) for experimental animals, or
aCSF only for control animals, and incubated overnight at
37.degree. C. prior to implantation. Following anesthesia as above,
and under sterile conditions, the 5 mm cannula attached to the
minipump (brain infusion kit, Alzet, Inc.) was stereotaxically
implanted into the left caudate-putamen (+1.2 A/P; +2.7 M/L) using
Bregma as a reference (Paxinos and Watson, 1986, supra) and fixed
to the skull with carboxylate cement (FIG. 1). The minipump itself
was placed subcutaneously in the interscapular region. The infusate
was delivered directly into the striatum over a period of two weeks
at a rate of 0.5 .mu.l/hour.
[0134] D. Tissue Preparation
[0135] At the end of the infusion period, animals were sacrificed
by decapitation. Their brains were quickly removed, frozen in
isopentane at -20.degree. C., and stored at -70.degree. C. Coronal
cryostat sections were cut at 20 .mu.m and thaw-adhered to
Vectabond.TM. (Vector Labs, Inc.) coated slides in ordered
anterior-to-posterior rows. The sections were postfixed with 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for one hour,
rinsed in phosphate buffer and air dried. Sections were-stored with
desiccant at -20 C. until processed.
[0136] E. Hybridization Probes
[0137] TGF.alpha. mRNA probes were generated from a 550 nucleotide
XbaI/BamHI cDNA fragment from the 5' end of rat TGF.alpha.
subcloned into pGEM 7Zf (Promega, Inc.). Antisense and sense probes
were transcribed with SP6 and T7 polymerases, respectively. Rat EGF
receptor mRNA probes were produced from a 718 base pair BamHI/SphI
insert from the 5' end of the gene in pGEM 7Zf. Probes for rat TH
were created using the 1.2 kb BamHI/EcoRI fragment subcloned into
pGEM 7Zf. Antisense subclones for EGF receptor and TH were
transcribed with T7 polymerase. Sense subclones for EGF receptor
and TH were transcribed with SP6 polymerase. All probes were
radiolabeled by transcription in the presence of [.sup.35S]UTP (NEN
Research Products, Inc.).
[0138] F. In situ Hybridization
[0139] In situ nucleic acid hybridization was performed according
to the method described by Simmons et al. 1989, supra). Parallel
sections from experimental and control animals were hybridized
overnight at 65.degree. C. with sense or antisense probes at a
concentration of 10.sup.7 cpm/ml. Adjacent sections from the same
animals were hybridized to each of the probes so that a direct
comparison could be made of their anatomical distributions.
[0140] Slides from experimental and control animals were grouped
together and apposed with .sup.14C-labeled brain paste standards to
autoradiographic BetaMax Hyperfilm.TM. (Amersham, Inc.) for three
to seven days. After successful development of the autoradiography
film, the slides were dipped in Kodak NTB-2 emulsion and exposed
for four weeks. The autoradiographic sheet film and NTB-2 emulsion
were developed with D-19 developer and Rapid Fix (Kodak, Inc.). The
brain sections were then counterstained with thionin and coverslips
were applied.
[0141] G. Analysis and Quantitation
[0142] Dipped and stained sections were examined and photographed
under bright and dark field microscopy. Autoradiograms were
analyzed quantitatively using an MCID system (Microcomputer Imaging
Device, Imaging Research, Inc.). Densitometry readings were sampled
at multiple sites within each anatomical region of interest and
averaged. Relative concentrations of TGF.alpha. and EGF receptor
hybridization were then estimated using a computer-generated third
degree polynomial standard curve constructed from the .sup.14C
brain paste standards. The estimated values for each region in each
treatment group were then averaged and their standard errors
calculated. Brain regions ipsilateral to the experimental
treatments were compared to the corresponding contralateral regions
in the same sections or to corresponding regions in control brains
at approximately the same positions. Significance of the
comparisons was determined using the Student's t-test.
[0143] H. Normal Expression and Control Infusions
[0144] Expression of TH, TGF.alpha. and EGF receptor mRNAs in the
striatum, the striatal subependymal region, and the SN in control
animals receiving striatal infusions of aCSF was indistinguishable
from that in normal animals (See Example 1 for normal developing
and adult expression). TH mRNA hybridization was prominent
throughout the SN-VTA. TGF.alpha. mRNA was not detected in the SN.
EGF receptor mRNA, however, was prominent in the medial substantia
nigra pars compacts (SNc), the paranigral, parabranchial, and
interpeduncular nuclei of the VTA (FIG. 2).
[0145] TGF.alpha. mRNA hybridization was expressed in the
caudate-putamen and nucleus accumbens (NA), being slightly less
dense in the NA. EGF receptor hybridization was found at low levels
throughout the body of the striatum and NA with higher punctate
expression dispersed throughout the low level background, and at
moderate levels in the proliferative regions of striatum bordering
the lateral ventricles.
[0146] I. Effects of 6-OHDA Lesions
[0147] Unilateral nigral 6-OHDA lesions reduced TGF.alpha. mRNA
hybridization in the ipsilateral striatum by 25%, but had no effect
on contralateral TGF.alpha. mRNA hybridization (FIG. 5). Striatal
and subependymal EGF receptor hybridization were unchanged in
lesioned animals compared to normal animals (FIG. 6). In the
midbrain, 6-OHDA lesions abolished EGF receptor hybridization in
the ipsilateral SN-VTA.
[0148] J. Effects of TGF.alpha. Infusions
[0149] In unlesioned animals receiving TGF.alpha. infusions,
TGF.alpha. mRNA hybridization in the infused striatum was unchanged
compared to the contralateral striatum or striata from normal
animals (FIG. 5). A few of the animals receiving infusions of
either TGF.alpha. or aCSF displayed a slight increase of TGF.alpha.
mRNA hybridization immediately around the infusion cannula scar.
TGF.alpha. mRNA hybridization in the substantia nigra was not
increased to detectable levels by the infusions.
[0150] Hybridization to EGF receptor mRNA was dramatically
increased in the ipsilateral subependymal region, but not in the
rest of the striatum, in all animals receiving TGF.alpha. infusions
(FIG. 6). No change from normal was observed in EGF receptor
hybridization in the SNc with TGF.alpha. infusion alone.
[0151] K. Combined TGF.alpha. Infusion and 6-OHDA Lesion
[0152] Striatal TGF.alpha. mRNA hybridization in animals receiving
both striatal TGF.alpha. infusions and subsequent nigral 6-OHDA
lesions was indistinguishable from that in lesion-only animals
(FIG. 5). Similarly, EGF receptor hybridization in the midbrains of
TGF.alpha.-infused/6-OHDA-- lesioned animals was indistinguishable
from that in lesion-only animals.
[0153] EGF receptor mRNA hybridization in the forebrain revealed an
anomalous ridge of dense hybridization in the body of the
ipsilateral striatum in addition to the increased hybridization in
the subependymal region (FIGS. 4, 6, and 7). The ridge was found in
five of the six rats in the combined TGF.alpha. infusion/lesion
group, but only in one of six rats in the
TGF.alpha.infusion/nonlesion group. EGF receptor hybridization in
the surrounding striatum was unchanged.
[0154] L. Discussion
[0155] The results described above permit analysis of the
modulatory effects of nigral 6-OHDA lesions or striatal infusions
of TGF.alpha., or both, on the expression of mRNAs encoding
TGF.alpha. and the EGF receptor in the adult rodent nigrostriatal
system. The results clearly demonstrated changes in expression
associated with each treatment individually and a unique pattern of
striatal expression when the two treatments were combined.
1. Effects of 6-OHDA Lesions
[0156] Midbrain lesions with 6-OHDA reduced TGF.alpha. mRNA
hybridization in the striatum by 25% at several weeks post-lesion.
If TGF.alpha. peptide levels parallel expression of TGF.alpha. mRNA
in this system, the decrease in TGF.alpha. mRNA may be one aspect
of the rodent lesion model that is not similar to idiopathic human
PD: TGF.alpha. is greatly increased in the striata of PD patients
(Mogi et al., Neurosci. Lett. 180:147-150, 1994). TGF.alpha. has
been shown to enhance a number of measure of dopamine neuron
function in vitro (Alexi and Hefti, Neurosci. 55:903-918, 1993).
The increase of TGF.alpha. (and EGF and other trophic factors) may
therefore reflect a response to the continuing degeneration of
dopamine neurons and their striatal efferents and may contribute to
the capacity of remaining midbrain dopamine cells to compensate for
the lost striatal dopaminergic innervation (Mogi et al., 1994,
supra). Thus, a partial- or perhaps a chronic-injury model might
better represent this aspect of human PD.
[0157] The difference in the time course of the loss of dopamine
cells may also help explain the apparent discrepancy between the
6-OHDA rodent model and human PD. In the rat model, midbrain
dopamine neurons are killed relatively quickly by a single
injection of neurotoxin. The chronic, progressive degeneration of
mesencephalic dopamine neurons in human PD occurs over many years.
In the present study, the animals were sacrificed well after
midbrain dopamine cells had degenerated. There may have been early
changes in striatal TGF.alpha. mRNA expression in these animals
that would not be apparent in our experiments. It would be of
interest to determine how TGF.alpha. mRNA expression varies in the
rat model over shorter periods post-lesion, while dopamine neurons
are in the process of degenerating.
[0158] Moderate TGF.alpha. mRNA expression in the caudate-putamen
is consistent with its putative role as a target-derived growth
factor for midbrain dopamine neurons. The underlying cause of its
decrease in the ipsilateral striatum after midbrain neurotoxic
lesion is unclear. Dopamine receptor binding has been shown to
influence TGF.alpha. mRNA expression in the hypothalamus
(Borgundvaag et al., Endocrinol. 130:3453-3458, 1992), but no such
interaction has yet been demonstrated in the striatum. TGF.alpha.
mRNA is expressed in subpopulations of neurons and glia in the
normal adult rodent striatum (Seroogy et al., J. Neurochem.
60:1777-1782, 1993). Dopamine denervation of the striatum could
potentially have influenced TGF.alpha. mRNA expression in
postsynaptic neurons, astrocytes or both.
[0159] Contralateral striatal TGF.alpha. mRNA expression was not
significantly altered by 6-OHDA lesion. This finding, too, is
consistent with a dopamine denervation-mediated decrease in
TGF.alpha. mRNA expression. Only a few percent of mesostriatal
dopaminergic projections are contralateral (Loughlin and Fallon,
Neurosci. Lett. 32:11-16, 1982), thus any contralateral regulatory
effects resulting from the lesion would be expected to be minor
compared to ipsilateral effects.
[0160] EGF receptor mRNA hybridization in the DA-denervated
striatum did not differ significantly from that in the
contralateral CP or in unlesioned control striata. Again, there may
have been early changes in expression due to the midbrain lesion or
implantation of the infusion cannula that were not manifest at
several weeks postlesion or two weeks post-implantation. The
abolition of EGF receptor mRNA hybridization in the lesioned SNc
confirms a similar observation after 6-OHDA lesion of the medial
forebrain bundle (Seroogy et al., Neuroreport 6:105-108, 1994).
This lesion-induced decrease was previously cited as evidence of
EGF receptor expression by nigral dopamine neurons (Seroogy et al.,
1994, supra), but the possibility remains of nigral glial
production of EGF receptor mRNA that is subject to regulation by
injury or death of nearby nigral dopamine neurons. Interestingly,
EGF receptor binding in the midbrains of postmortem PD patients is
unchanged from normals (Villares et al., Brain Res. 628:72-76,
1993). Thus, the loss of EGF receptor mRNA expression after lesion
represents another difference between the rodent lesion model and
human PD. As with TGF.alpha. expression in the striatum, a
partial-lesion model may better mimic in a rat brain the changes
seen in a Parkinsonian human brain.
2. Effects of TGF.alpha. Infusions
[0161] In unlesioned animals, infusion of TGF.alpha. or aCSF did
not significantly alter TGF.alpha. mRNA hybridization from normal
levels in the midbrain or striatum. Despite reports of
autostimulation of TGF.alpha. expression in other tissues or cell
types (Coffey et al., Nature 328:817-820, 1987; Barnard et al., J.
Biol. Chem. 269:22817-22822, 1994), results from the present study
do not provide evidence for such activity in this system. The
autostimulatory effects in those earlier studies were produced on
the order of a few hours. Brain tissue in the present study was
obtained after continuous exposure to the growth factor over a
period of two weeks. Thus, an early upregulation near the beginning
of the infusion that later subsided would not be evident.
[0162] As with TGF.alpha. and EGF receptor transcripts in
lesion-only animals, it would be of interest to examine the time
course of modulation at time points earlier after onset of the
experimental treatment. The slight increase in TGF.alpha. mRNA seen
in a few animals immediately around the infusion scar was found in
both TGF.alpha.- and aCSF-infused striata. Thus, it is probably
attributable to continued mechanical injury and gliosis caused by
the cannula itself, and not to the infusate.
[0163] TGF.alpha. infusions dramatically increased EGF receptor
mRNA hybridization in the ipsilateral subependymal zone but not in
the rest of the striatum. In other tissues, EGF receptor mRNA can
be modulated by several chemical and mechanical means. EGF peptide
increased EGF receptor mRNA in numerous mammalian cell types in
vitro (Earp et al., J. Biol. Chem. 261:4777-4780, 1986; Kesavan et
al., Oncogene 5:483-488, 1990). Retinoic acid caused a similar
increase in normal rodent fibroblasts (Thompson and Rosner, J.
Biol. Chem. 264:3230-3234, 1989) and in a transformed rat liver
cell line (Raymond et al., Cell. Growth Diff. 1:393-399, 1990).
Exposure to cyclohexamide, by itself or with EGF, stimulated an
increase in cultured human cytotrophoblasts and stabilized EGF
receptor transcripts, thus providing a mechanism other than
enhanced transcription to increase total abundance of EGF receptor
mRNA (Kesavan et al., 1990, supra).
[0164] Transection of the sciatic nerve in rats brought about a
graded increase in EGF receptor mRNA in the severed ends (Toma et
al., J. Neurosci. 12:2504-2515, 1992). Treatment with protamine
increased .sup.125I-EGF binding and cell surface receptor number in
mouse and human cell lines in vitro (Lokeshwar et al., J. Biol.
Chem. 264:19318-19326, 1989). TGF.alpha. may mimic these actions
and increase the production and/or longevity of EGF receptor
transcripts in extant subependymal cells. It may also stimulate
transcription in cells that do not normally express appreciable
amounts of EGF receptor mRNA. Both of these effects could be
readily investigated further in vivo with routine techniques known
to those of ordinary skill in the art.
[0165] An additional possibility is that TGF.alpha. is stimulating
proliferation of subependymal cells and increasing the total
numbers of cells expressing EGF receptor mRNA. EGF and TGF.alpha.
are potent mitogens for cultured "EGF responsive" cells explanted
from the subependymal region (Reynolds and Weiss, 1992, Science
255:1707-1710). The strong inductive effect striatal TGF.alpha.
infusion had on subependymal EGF receptor mRNA hybridization may
indicate that proliferative cells in the intact brain respond
similarly to these cell in vivo.
3. Combined TGF.alpha. Infusion and 6-OHDA Lesion
[0166] In animals receiving combined lesions and TGF.alpha.
infusions, striatal TGF.alpha. mRNA hybridization was
indistinguishable from that in animals receiving combined lesions
and aCSF infusions. Although TGF.alpha. has potent autostimulatory
effects in other tissues, it did not significantly alter the
reduction of striatal TGF.alpha. mRNA hybridization in the present
study. The 6-OHDA-mediated loss of mesencephalic EGF receptor mRNA
was similarly unaffected by TGF.alpha. infusion. In the latter
case, the midbrain lesions were performed, and ipsilateral dopamine
cells destroyed, weeks prior to the start of the infusion. Thus,
the growth factor would not have had an opportunity to prevent
their elimination.
[0167] There is some evidence that the dopamine cells themselves
express EGF receptor mRNA (Seroogy et al., 1994, supra) and that
TGF.alpha. can moderate the loss of markers for striatal
dopaminergic innervation if administered concurrently with the
neurotoxin. Therefore, the time interval between the neurotoxic
lesions and the administration of TGF.alpha. may explain why
TGF.alpha. infusions had no impact on the abolition of midbrain EGF
receptor mRNA.
[0168] In human Parkinsonian brains, mesencephalic EGF receptor
binding is unchanged from that seen in the normal brain (Villares
et al., 1993, supra). The huge increases in striatal TGF.alpha.
(and other neurotrophic factors) with PD (Mogi et al., 1994, supra)
may mask a reduction in the number of EGF receptor expressing
dopamine cells by increasing the levels of expression in the
remaining neurons. On the other hand, in vitro experiments suggest
that many of the trophic effects of TGF.alpha. on mesencephalic
dopamine neurons are mediated, at least partially, through glia
(Alexi and Hefti, 1993, supra). TGF.alpha. may therefore act
through paracrine (direct) and sequential (indirect) modes of
transport to influence dopamine neurons.
[0169] The pattern of EGF receptor mRNA hybridization in the
subependymal zone of TGF.alpha.-lesioned animals was similar to
that seen in TGF.alpha.-nonlesioned animals. The most striking
feature in the ipsilateral striata of these animals was a dense
ridge of hybridization well out of the body of the striatum, more
intense even that the enhanced hybridization in the subependymal
zone. The ridge did not correspond to any known anatomical feature
and was not evident with the TGF.alpha. or TH probes. EGF receptor
mRNA hybridization in the non-ridge striatum was the same as in the
striata of all other groups.
[0170] The neurotoxic damage from the 6-OHDA lesions and the
mechanical injury from implantation of the infusion cannula may
have stimulated proliferation and activation of glial cells.
Previous studies have demonstrated gliosis and increased astrocytic
EGF receptor expression as a result of injury (Nieto-Sampedro et
al., Neurosci. Lett. 91:276-282, 1988; Fernaud-Espinoza et al.,
Glia 8:277-291, 1993). Further, TGF.alpha. may play a role in the
reactivity of astrocytes (Junier et al., J. Neurosci. 14:4206-4216,
1994).
[0171] Another possibility is that proliferative cells of the
subependymal region were drawn away from the ventricle and into the
overlying striatum by the combined growth factor infusion and
midbrain lesion. TGF.alpha. is a potent chemoattractant for diverse
cell types (Reneker et al., Development 121:1669-1680, 1995), but
by itself was not sufficient in most animals to stimulate formation
of the ridge. Formation of the cellular ridge may have been
facilitated by the midbrain lesions. The origin and identity of
these cells will be examined in the following example.
Example 3
Characterization of the Striatal Ridge
[0172] As described above, striatal infusions of TGF.alpha., when
combined with nigral 6-OHDA lesions, induce the formation of a
dense ridge of cells in the body of the striatum that abundantly
expresses EGF receptor mRNA but no more TGF.alpha. than the
surrounding tissue. The ridge was comprised of a mass of densely
packed cells, allowing its clear detection using simple thionin
staining. The identity of the anomalous striatal ridge was not
apparent, but three possibilities were considered.
[0173] Gliosis in response to injury is a feature of both traumatic
and neurotoxic damage to brain tissue. Typically, both types of
brain injury stimulate astrocytosis and infiltration of injured
tissue by astrocytes and microglia (Fernaud-Espinoza et al., Glia
8:277-291, 1993). Astrocytes have been shown to express EGF
receptor immunoreactivity, particularly in response to brain injury
(Gomez-Pinilla et al., Brain Res. 438:385-390, 1988; Nieto-Sampedro
et al., Neurosci. Lett. 91:276-282, 1988). In addition, TGF itself
stimulates the proliferation of astrocytes (Alexi and Hefti,
Neurosci. 55:903-918, 1993). Therefore, the possibility that the
striatal ridge was a mass of glial cells responding to the combined
neurotoxic and mechanical damage and infusion of the growth factor
was considered.
[0174] A second potential source for the ridge was investigated
that was related to the distinctive anatomy of the rodent striatum.
The ridge did not correspond to any previously identified
anatomical feature. In rodents, the caudate and putamen are not
anatomically distinct structures. No anatomical or neurochemical
markers have been identified thus far that distinguish between
these two nuclei of the basal ganglia in rodents. However, during
prenatal and early postnatal development, neurogenetic gradients
within different regions of the developing striatum correspond to
characteristic gradients in the caudate and putamen in animals
where these nuclei are anatomically discrete (Bayer, Intl. J.
Devel. Neurosci. 2:163-175, 1984). In rodents, new striatal neurons
rostral to the decussation of the anterior commissure are added in
a lateral-to-medial gradient such that the latest born neurons are
those nearest the lateral ventricle. That same pattern is observed
in the development of the caudate nucleus, suggesting that the
anterior striatum in rodents is more of a "caudate-like" region.
Caudal to the crossing of the anterior commissure, neurons are
added in a medial-to-lateral gradient, similar to the developing
putamen in animals where it is anatomically distinct. Thus, the
posterior rodent striatum may be more "putamen-like". The
possibility was considered that the striatal ridge, then, might
represent a previously unrecognized border between these two
regions of rat striatum that allowed a dense buildup of cells,
perhaps due to some neurochemical difference.
[0175] A third possibility for the source of the striatal ridge was
also examined. Explanted cells from the subependymal zones of the
forebrain lateral ventricles of adult mammals have been found
capable of proliferating and differentiating into new neurons and
glia, particularly when cultured in the presence of EGF-family
neurotrophic factors, including TGF.alpha. (Reynolds et al., J.
Neurosci. 12:4565-4574, 1992; Morshead et al., Neuron 13:1071-1082,
1994). Recently, EGF or TGF.alpha. infused into the lateral
ventricle stimulated proliferation of "EGF-responsive" stem and
neural progenitor cells in the adult mouse brain (Craig et al., J.
Neurosci. 16:2649-2658, 1996). A possibility for the source of the
striatal ridges in the present studies was that the TGF.alpha.
infusions stimulated similar proliferative activity in the brains
of rats. Additionally, the possibility that the striatal ridges
were mass migrations of proliferating neural progenitor cells
derived from the subependymal regions was considered.
[0176] The experiments described below served to allow
characterization of this anomalous striatal ridge using a variety
of histochemical and immunohistochemical techniques. The origin of
the ridge and the factors influencing its appearance were also
investigated by examining the time course of its formation in the
striatum and by altering the combinations of surgical and chemical
treatments.
[0177] A. Experimental Protocols
[0178] Adult male Sprague-Dawley rats weighing 250-300 grams were
used throughout the study. Twenty-four animals received standard
midstriatal infusions of rat TGF.alpha. (0.5 .mu.g/day; Sigma
Chemical Co.). Another 26 rats received either artificial
cerebrospinal fluid (aCSF) or no infusion. A subset of animals in
the standard TGF.alpha. infusion group and the control group
received stereotaxic 6-OHDA injections into the substantia nigra 48
hours after the infusions were begun. Animals used in this portion
of the study were classified into six groups according to their
infusion-lesion combination, as follows: TGF.alpha. infusion,
lesion (n=13); TGF.alpha. infusion, no lesion (n=11); aCSF
infusion, lesion (n=12); aCSF infusion, no lesion (n=9); no
infusion, lesion (n=1); no infusion, no lesion (n=4).
[0179] Additional animals received TGF.alpha. infusions into other
regions of striatum, the lateral ventricle, cerebral cortex, or the
septum. Four more animals (two per group) received midstriatal
TGF.alpha. infusions at one-half or one-tenth the standard dose.
Also, two animals received midstriatal infusions of epidermal
growth factor (EGF) instead of TGF.alpha.. The EGF administered in
these rats was at the standard 0.5 .mu.g/day dose. All of the
animals in these extra groups were lesioned. Rats in all of the
experiments were typically perfused one to 16 days postlesion (3-18
days of infusion). To determine whether the ridge would persist
after the infusions ceased, the minipumps were removed from four
animals with TGF.alpha. infusions at the end of two weeks, but
these animals were not perfused until several days later. Brain
sections were prepared and stained using various immunocytochemical
and histochemical techniques.
[0180] B. TGF.alpha. Infusion
[0181] Rats were anesthetized with 8 mg xylazine and 100 mg
ketamine/kg. Infusions of TGF.alpha. were provided up to 18 days by
Alzet osmotic minipump (2002). The minipumps were filled to about
200 .mu.l with either aCSF for control animals or 20 .mu.g
TGF.alpha. in 400 .mu.l of aCSF (50 .mu.g/ml) for experimental
animals. Under sterile conditions, the infusion cannula was
positioned to stereotaxic coordinates (+1.2 A/P; +2.7 M/L; -6.0
D/V) based on Bregma (Paxinos and Watson, The Rat Brain in
Stereotaxic Coordinates, Academic Press, San Diego, 1986) and
cemented to the top of the skull with dental cement. The infusate
was delivered via cannula at approximately 0.5 .mu.l/hour. Some
additional control animals received infusions either into the
lateral ventricle, the overlying cortex, or other areas.
[0182] C. Neurotoxic Lesion
[0183] Forty-eight hours after the minipump implant, rats were
anesthetized as above. A chilled 4.8 mg/ml solution of 6-OHDA HCl
was prepared immediately prior to injection. Using sterile
technique, the neurotoxin was stereotaxically injected into the
ipsilateral substantia nigra (+3.7 A/P; +2.1 M/L; +2.0 D/V) using
interaural zero as a reference (Paxinos and Watson, 1986, supra). A
6-8 .mu.l volume was injected at a rate of 1 .mu.l/minute.
[0184] D. Tissue Preparation
[0185] Animals were perfused with 500 ml of 4% paraformaldehyde in
0.1 M phosphate buffered saline (PBS; pH 7.4) one to 16 days
postlesion and their brains placed into 20% sucrose. The next day,
the brains were frozen in isopentane at -20.degree. C. Forty-micron
coronal sections were then cut on a freezing microtome into 2%
paraformaldehyde in 0.1 M PBS. Continuous sections were taken
through the striatum and substantia nigra-VTA. Representative
sections were taken through the rest of the brain.
[0186] E. Nissl Staining
[0187] Microtomed brain sections for Nissl staining were mounted
onto gelatin-coated slides and allowed to dry overnight. They were
then dehydrated and rehydrated through ethanol baths, and placed in
Thionin solution for approximately four minutes. The sections were
dehydrated through the series of ethanol baths, cleared in
successive Histoclear washes, and coverslipped. Section were viewed
under light microscopy and photographed with Technical Pan Film
(Kodak, Inc.) at ISO 100 (HC-110 processing for six minutes).
[0188] F. Silver Staining
[0189] Degenerating fibers and cells of the ridge were labeled
using a modification to the Nauta staining method, similar to
Procedure I of Fink-Heimer (Giolli and Karamanlidis, In:
Neuroanatomical Research Techniques, R. T. Robertson, Ed., pp.
211-240, Academic Press, New York, 1978). Briefly, free-floating
sections were placed into 0.05% potassium permanganate prior to
treatment with fresh 1% hydroquinone-1% oxalic acid. They were
treated with successive uranyl nitrate/silver nitrate solutions of
increasing concentration. After another rinse, the sections were
reacted in ammoniacal silver, then in ethanol/citric
acid/paraformaldehyde reducer, and finally in sodium thiosulfate.
After staining, the sections were mounted on glass slides and
allowed to dry on a slide dryer for 15 minutes. The sections were
then dehydrated through successive ethanol washes of increasing
concentration, defatted in three successive Histoclear washes, and
coverslipped.
[0190] G. Immunohistochemistry
[0191] The quality and the extent of the nigral lesion were
determined by the loss of TH-IR in the ipsilateral ventral
midbrain. Antibodies against glial fibrillary acidic protein
(GFAP), a marker for astrocytes, nestin, a marker for neural
progenitor cells, and vimentin, a marker for radial glial cells,
were employed in the neurochemical characterization of the ridge.
Immunohistochemistry was performed on free-floating sections.
Briefly, brain sections were washed in 0.1 M PBS or Tris buffered
saline (TBS; 3.times.10 minutes) then incubated for 1 hour in
blocking solution consisting of 3% normal goat serum in 0.1 M PBS
or TBS with 250 .mu.l Triton X-100. Next, they were incubated
overnight at room temperature on a rotator with antibody solution
diluted with blocking solution: rabbit anti-TH antiserum (1:500;
Eugene Tech Intl., Inc.), rabbit anti-GFAP (1:6400; Dako Corp.),
mouse monoclonal anti-vimentin (1:50; Sigma Chemical Co.), or with
mouse anti-nestin supernatant (1:20; University of Iowa Hybridoma
Bank).
[0192] The sections then were washed and incubated for 1 hour with
biotinylated goat anti-rabbit antiserum (1:200; Vector Labs, Inc.)
for TH or GFAP immunostaining, or biotinylated horse anti-mouse
antiserum (Vector Labs, Inc.) for nestin or vimentin
immunostaining, then washed and incubated in avidin-biotin complex
(ABC Elite kit, Vector Labs, Inc.) for 1 hour. Localization of
primary antibody binding was revealed using the diaminobenzidine
(DAB) peroxidase technique. The sections were washed thoroughly and
mounted on gelatin-subbed slides and allowed to dry overnight.
Finally, the sections were dehydrated, cleared, and coverslipped as
described above.
[0193] None of the animals used in the study displayed adverse
effects from the minipump implants or lesion surgeries. All
continued to take food and water through the course of the
experiments. If the lesion, infusion, or both were not successful,
the animals (n=6) were excluded from the initial experimental
groups and examined separately. A successful lesion was defined as
one that caused complete or near-complete elimination of
ipsilateral nigral TH-IR. A successful striatal infusion was
defined as one where the tip of infusion cannula was successfully
fixed into the body of the striatum.
[0194] As with the in situ hybridization studies, all animals
receiving intrastriatal TGF.alpha. infusions of six days or more
displayed a dramatic buildup of cells along the ventricle
ipsilateral to the infusion, visible with thionin staining. By
comparison, the contralateral striatum showed no such increase, and
was indistinguishable from that in aCSF-infused animals. EGF
infusions in lesioned animals induced the cellular expansion along
the ventricle, but did not induce formation of the striatal ridge.
Lower doses of TGF.alpha. induced both the cellular expansion and
ridge formation, but, qualitatively, the number of cells in each
was decreased.
1. Effects of 6-OHDA Lesions
[0195] None of the lesioned animals receiving aCSF infusions showed
any cellular expansion along the ipsilateral ventricle or any
evidence of a striatal ridge. Lesioned animals infused with
TGF.alpha. did uniformly exhibit the buildup of cells along the
ventricle and typically displayed the striatal ridge. Nigral
lesions dramatically increased the incidence of formation of the
ridge compared to unlesioned animals (Table 1).
2. Morphology and Persistence of the Ridge
[0196] Midstriatal infusion resulted in a characteristic S-shaped
ridge arising from the dorsomedial caudate-putamen, sweeping out
into the striatum and looping back slightly toward the midline at
its ventral end. The dorsal-most portion of the ridge was
continuous with the build-up of cells in the subependymal region.
Typically, thionin staining was most dense in the dorsal portion of
the ridge paralleling the EGF receptor mRNA hybridization. The
cellular ridge was generally found throughout most of the
rostral-caudal extent of the striatum. The ridge was still
prominent in the striatum three months after the TGF.alpha.
infusion pump was removed.
1 Infusate Lesion Number Expansion aCSF no 9 0% 0% aCSF yes 12 0%
0% TGF.alpha. no 11 100% 27% TGF.alpha. yes 13 100% 92%
3. GFAP Immunohistochemistry
[0197] Antiserum against glial fibrillary acidic protein (GFAP), a
marker for astrocytes, failed to stain cells of the striatal ridge
or the cellular expansion along the ventricle. Normal GFAP-IR
astrocytic staining was found medial and lateral to the ridge, but
was nearly excluded from the ridge itself.
4. Silver Staining
[0198] Labeling cells non-specifically with a modification of the
Nauta method provided additional information about the cells
comprising the ventricular cellular expansion and the striatal
ridge. One of the most striking features was the huge number of
cells making up the subependymal cellular buildup and ridge (FIG.
8). The cells were densely packed and predominantly fusiform in
shape (FIG. 8). In the ventral portion of the ridge, elongated
cells appeared to stream around fiber bundles of the internal
capsule suggesting that the cells were migrating through the
striatum.
5. Time Course of Ridge Formation
[0199] The density of cells of the ridge allowed us to track its
formation using simple thionin staining (FIG. 9). All animals used
for the time-course experiment received 6-OHDA lesions and
midstriatal TGF.alpha. infusions. At time points prior to six days
of infusion, there was only a very minor build-up of cells in the
subependymal region and no evidence of a striatal ridge. By six
days of infusion, there was a clear expansion of cells along the
ventricle. At nine days infusion, the ventral portion of the ridge
had begun to appear slightly displaced from the ventricle. By 12
days infusion, the ventral portion of the ridge was situated as
much as 400 .mu.m from the ventricle wall. At 16 days infusion, the
ridge appeared midstriatum, its ventral portion up to two mm from
the ventricle. Thus, the ridge originated in the ventricular region
and was increasingly displaced radially in the overlying striatum
at greater times of infusion. The estimated difference in distance
between the lateral extents of the ridges and the ventricle wall at
12 days and 16 days of infusion was approximately 1.6 mm.
6. Nestin Immunohistochemistry
[0200] Monoclonal antibodies against nestin, a marker for
neuroepithelial progenitor cells, intensely stained dense
collections of fibers throughout the ridge (FIG. 10). No nestin-IR
fibers were seen lateral to the ridge, but occasional fibers were
observed medial to the ridge. The fibers were oriented primarily
orthogonal to the ridge.
7. Alteration of Ridge Morphology
[0201] Lesioned animals infused midstriatally with TGF.alpha.
uniformly exhibited a characteristic S-shaped striatal ridge in
coronal sections. This morphology was dramatically altered in rats
with infusions into other areas of the caudate-putamen (FIG. 11).
Medial striatal infusions gave rise to an L-shaped ridge near the
cannula infusion site with the vertical part of the "L" along the
ventricle and the horizontal part extending orthogonally from the
ventricle into the striatum. Infusion into the extreme lateral
striatum stimulated the formation of a linear ridge parallel to the
wall of the lateral ventricle.
8. Vimentin Immunohistochemistry
[0202] Antiserum recognizing vimentin, a marker for radial glial
cells, failed to stain any cells in the striatum, the subependymal
zone, or the striatal ridge at two weeks of infusion. However, this
result is being investigated further, as the controls were
performed in embryonic animals, under conditions that may not
ensure elimination of false negatives.
9. Control of Ridge Position
[0203] Compared to the cells of ridges in rats used for in situ
hybridization experiments, ridge cells in the present series of
experiments were maximally displaced much farther from the
ventricle (FIG. 12). The difference between these two groups of
animals experimentally was the timetable of infusions and
lesions.
[0204] Animals prepared for in situ hybridization experiments
received lesions and then underwent a series of behavioral tests
starting around the second week postlesion to confirm success of
the unilateral lesions. Typically, those animals did not receive
infusions until five weeks after the lesion. Thus, the dopamine
degenerative process and the striatal infusion of TGF.alpha. were
temporally separate events.
[0205] In the present series of experiments, infusions were begun
first; lesions were not performed until 48 hours after the infusion
pumps were implanted. In these animals, the degeneration of the
nigral dopamine neurons, the resulting loss of striatal
dopaminergic innervation, and the striatal administration of growth
factor were temporally concurrent events.
10. Infusions Into Brain Regions Other Than the Striatum
[0206] All rats receiving infusions into brain areas other than the
striatum received two-week TGF.alpha. infusions and nigral 6-OHDA
lesions. Intracerebroventricular (ICV) infusion of growth factor
ipsilateral to the lesion stimulated the buildup of cells in the
adjacent ventricular wall, but did not induce formation of the
striatal ridge in any of the animals.
[0207] Septal and some striatal infusions stimulated the formation
of septal ridges associated with the medial walls of the lateral
ventricles. Septal ridges, like the striatal ridges, were readily
detected with EGF receptor mRNA in situ hybridization or with
thionin staining, but tended to be qualitatively less robust in
terms of the density and number of cells.
[0208] Dorsal cortical infusions, that is, infusions so shallow
that they did not penetrate the corpus callosum, had no discernable
effect on cell density along the lateral ventricle. Neither did
these dorsal infusions induce formation of a ridge. Cortical
infusions in which the corpus callosum was slightly penetrated
stimulated expansion of cells along the ipsilateral ventricle, but
did not induce formation of a striatal ridge. These animals did
exhibit densities of cells in the corpus callosum that might be
considered callosal ridges.
H. TGF.alpha. Stimulates Cellular Proliferation
[0209] The present experiments, together with those described
above, demonstrate that TGF.alpha. administration was necessary for
the formation of the cellular build-up and the striatal ridge. Not
a single animal that received a striatal aCSF infusion--whether
lesioned or not--displayed any obvious periventricular cellular
expansion when compared to subependymal regions contralateral to
the infusions or to normal animals. Clearly, cells of the forebrain
are responding to striatal infusion of TGF.alpha. by proliferating
along the lateral ventricle.
[0210] Recent studies with 6-day ICV infusions of EGF or TGF.alpha.
in mice demonstrated a large increase in the number of cells around
the ventricle immunolabeled with 5-bromo-2'-deoxyuridine (BrdU) or
[.sup.3H]thymidine, markers for cellular proliferation (Craig et
al., J. Neurosci. 16:2649-2658, 1996). More than 95% of these cells
were also positively immunoreactive for EGF receptor. Cresyl violet
Nissl staining also showed an increase in the numbers of cells
around the ventricles in these animals in response to growth factor
administration.
[0211] The possibility that the expanded cell populations along the
lateral ventricles were glial cells stimulated by the combined
neurotoxic lesion and mechanical injury of the forebrain, and the
infused TGF.alpha. was considered first. Astrocytes are known to
respond to brain injury by proliferating and altering their
morphology and functional properties (for review see Norenberg, J.
Neuropath. Exper. Neurol. 53:213-220, 1994). Additionally, striatal
astrocytes possess EGF receptors (Gmez-Pinilla et al., 1988, supra;
Nieto-Sampedro et al., 1988, supra) and are stimulated by
TGF.alpha. to proliferate (Alexi and Hefti, 1993, supra). In the
present study, antiserum against glial fibrillary acidic peptide
(GFAP), a marker for astrocytes, failed to demonstrate an increase
in astrocytes in the ventricular region or in the ridge at two
weeks of infusion. In fact, GFAP-IR was largely excluded from these
areas. Normal astrocytic staining was seen medial and lateral to
the ridge, for instance, but few GFAP-IR fibers were observed
within the ridge itself. These findings paralleled those in
experiments with six-day ICV infusions of EGF or TGF.alpha.: GFAP
and an additional marker for astrocytes, S100.beta., showed no
significant increase around the lateral ventricle (Craig et al.,
1996, supra). Vimentin can also be expressed by reactive astrocytes
(Federoff et al., J. Neurosci. Res. 12:14-27, 1984), but
vimentin-IR was not observed in any of the sections examined.
Markers for microglia (MAC-1) and oligodendrocytes (MAG, CNP, O4
and Rip) also did not change significantly (Craig et al., 1996,
supra). Thus, the immunohistochemical evidence demonstrated that
the TGF.alpha.-induced expansion of cells along the ventricle and
in the striatal ridge were not the result of gliosis.
1. Cellular Morphology and Orientation
[0212] Silver and thionin staining clearly reveal the huge numbers
of cells within the cellular aggregation along the ventricle. The
cells were most dense and most numerous in the dorsal portions of
the subependymal zone and ridge. The cells were predominantly
fusiform, similar to migrating neural progenitor cells in the
developing brain, with their long axes oriented orthogonal to the
ventricle wall (or to the dorsolateral extension of the
subependymal zone bordering the dorsal striatum). Silver stained
cells in the ventral segment of the ridge appeared to stream around
fiber bundles of the internal capsule, suggesting that they were
migrating through the striatum.
[0213] To determine whether the cells were indeed migrating, a time
course experiment was performed to examined the development of the
ridge and its location as a function of time after the start of the
growth factor infusion.
2. Migration of Cells of the Striatal Ridge
[0214] The progressive expansion of cells along the lateral
ventricle and the subsequent radial movement of these cells as a
dense ridge proved that the cells were indeed migrating en masse
through the striatum. As such, the ridge could not have been an
anatomical delineation between the rodent putative "caudate-like"
and "putamen-like" regions of striatum.
3. Stimulation of Neural Precursor Cells
[0215] Although none of the immunomarkers for mature astrocytes,
neurons, microglia, or oligodendrocytes labeled cells of the
periventricular expansion or the striatal ridge, monoclonal
antibodies recognizing nestin, an intermediate filament expressed
by neuroepithelial precursor cells, intensely stained cell
processes in the ridge and along the ventricle. Reactive astrocytes
can also express nestin-IR, but the negative GFAP-IR in cells of
the ridge eliminated the possibility that astrocytes formed a
significant portion of the ridge cells. Nestin-IR has been used in
recent years to identify and label neural precursor cells in vitro
and in vivo (Lendahl et al., Cell 60:585-595, 1990; Craig et al.,
1996, supra). The strong nestin-IR in the striatal ridge and the
lack of immunostaining for glial markers support the conclusion
that the cells of the ridge are predominantly neural progenitor
cells.
[0216] Thus far, two distinct cell populations have been identified
in adult mammalian brain that can give rise to new neurons and glia
(Morshead et al., 1994, supra). One, the relatively quiescent cell
population, are believed to be true multipotent neural stem cells.
The other, the constitutively proliferating population, are
believed to be neural progenitor cells, descendent from the stem
cells. The stem cell population is thought to remain in the
ependymal or subependymal zone and replenish the progenitor cell
population as they die or migrate away. The term "neural precursor"
is used here to describe any undifferentiated proliferative cell
capable of giving rise to neurons and glia in the adult mammalian
brain, whether these cells are neural stem cells or neural
progenitors.
[0217] Previous studies have shown that many neural progenitor
cells die in the subependymal zone before they can migrate from the
region (Morshead and Van der Kooy, J. Neurosci. 12;249-256, 1992).
However, it was recently discovered that many others indeed survive
and migrate along a highly-restricted path to the olfactory bulbs
where they differentiate into olfactory interneurons (Luskin,
Neuron 11:173-189, 1993; Lois and Alvarez-Buylla, Science
264:1145-1148, 1994). They migrate tangentially along the wall of
the lateral ventricle in a process called "chain migration" wherein
chains of migrating cells are ensheathed by specialized GFAP-IR
astrocytes (Rousselot et al., 1994; Lois et al., Science
271:978-981, 1996).
[0218] The subependymal zone along the forebrain lateral
ventricles, then, is much more than a dormant remnant of the
embryonic neuroepithelium. In normal unmanipulated brains, it
continues to give rise to new neuroblasts that migrate rostrally
and differentiate into neurons. Under the influence of EGF-family
neurotrophic factors, including TGF.alpha., subependymal neural
precursors can be stimulated in vitro to give rise to large numbers
of new neurons, astrocytes and oligodendrocytes (Reynolds and
Weiss, Science 255:1707-1710, 1992). From these explant studies, it
became clear that the highest concentrations of neural precursor
cells were found in the dorsal portion of the subependymal zone,
along the dorsal border of the caudate-putamen.
[0219] There is even some recent evidence that neural precursors
may be stimulated to increase their numbers and produce new neurons
and glia in vivo (Craig et al., 1996, supra). Cells double-labeled
with BrdU and markers for mature neurons and glia were found
diffusely distributed throughout the striatum, septum, and cortex
after six days of ICV infusion of EGF and up to seven weeks of
post-infusion survival. However, no mass migration of subependymal
cells into the adjacent striatum was observed in that study (in
marked contrast to the results obtained by the present method) and
the numbers of cells were quite modest compared to the
densely-packed mass of cells observed in the striatal ridge
described herein. Moreover, none of the animals in Craig et al.
received an infusion sufficient to stimulate the mass migration
presently observed and none of the animals in that study received
nigral 6-OHDA lesions, which are shown for the first time herein to
dramatically increase the incidence of migration.
4. Evidence of Neuronal Phenotype
[0220] A question that remained was whether the cells within the
massive expansion along the ventricle and the migrating striatal
ridge truly were neural progenitor cells. Table 2 summarizes the
data supporting the conclusion that these cells are indeed neural
progenitors. The neurochemical evidence showed that they do not
express markers for mature neurons, astrocytes, oligodendrocytes,
or microglia. They did, however, intensely express a marker for
immature neural progenitor cells. They expressed markers for
cellular proliferation. They arose from the wall of the lateral
ventricle where neural precursors are located in the adult rodent
brain, expanded laterally and migrated radially away from the
ventricle. Furthermore, their cellular morphology was fusiform and
their processes were oriented orthogonal to the ventricle and the
ridge, similar to migrating neural progenitors in the embryonic
brain and consistent with their migration from the subependymal
region. The data summarized in the following Table (Table 2)
indicates that cells of the periventricular expansion and the
striatal ridge are neural progenitors arising from subependymal
neural stem cells.
2 Evidence of Progenitor Phenotype for Cells of the Expansion and
Ridge Neurochemical Express abundant EGF receptor mRNA* and
immunoreactivity Immunonegative for GFAP* or S-100.beta. markers
for astrocytes Immunonegative for NeuN, a marker for mature neurons
Immunonegative for MAG, CNP, 04, or Rip, markers for
oligodendroctyes Immunonegative for vimentin*, a marker for radial
glia Immunonegative for MAC-1, a marker for microglia
Immunopositive for nestin*, a marker for neuroepithelial precursors
Morphological Elongated somata in ridge oriented orthogonal to
subependymal zone Nestin-IR processes in ridge oriented normal to
subependymal zone Anatomical Arise from the subependymal zone
Highest density of cells is in the dorsal subependymal region
Physiological Respond to TGF.alpha. administration by increasing
their numbers
5. Mechanisms of Migration
[0221] The mass migration of these cells into the striatum could be
controlled by striatal dopamine denervation and by the location of
the infusion cannula, but the mechanism of migration was unclear.
The fact that the shape of the ridge could be modified simply by
changing the site of infusion initially suggested a chemoattractant
effect. TGF.alpha. is known to be a potent chemoattractive agent
for diverse cell types (Ju et al., J. Invest. Dermatol.
100:628-632, 1993; Panagakos, Biochem. Mol. Biol. Int. 33:643-650,
1994). Its abundant expression in the perinatal caudate-putamen may
indicate that it performs a similar role in the development of the
striatum.
[0222] Neural precursor cells in the normal brain are located in a
thin region in the wall of the lateral ventricle. Infusions into
the mid-striatum were closer to cells of the dorsal end of this
region. Presumably, cells migrating into the striatum would move
toward putative higher concentrations of growth factor at the tip
of the infusion cannula where the TGF.alpha. was released. The
characteristic S-shape of ridges in animals with mid-striatal
infusions might have resulted from receptor saturation of cells in
the dorsal segment of the subependymal zone nearest the tip of the
infusion cannula. Cells with saturated EGF receptors might have
halted their migration once they moved close to the infusion site.
Cells near the ventral end of the subependymal zone would see a
lower putative growth factor concentration and would have to travel
farther toward the infusion site before the concentration of
TGF.alpha. increased enough to saturate their receptors. This
differential migration with receptor saturation could explain the
characteristic S-shape of these ridges.
[0223] Infusions into the medial striatum resulted in L-shaped
ridges, again in keeping with a neurochemical gradient/receptor
saturation effect. In this instance, dorsal subependymal cells may
have had their receptors saturated and their migration halted
before they could even emerge from the subependymal zone. Only
cells in the most ventral portion of the subependymal region could
migrate away from the ventricle.
[0224] Extreme lateral infusions essentially would have presented a
similar putative concentration gradient to cells along most of the
length of the proliferative region. The subepenpdymal cells all
migrated a similar distance from the ventricle, resulting in a
roughly linear ridge, consistent with the idea of a
chemoattractant, neurochemical gradient effect.
[0225] However, immunohistochemical evidence from the
characterization studies cast doubt on the idea that a simple
chemoattractant effect could entirely explain the mass radial
migration. Nestin-IR processes of the migrating cells were not
aligned with the tip of the infusion cannula, the region of the
putative highest concentration of growth factor. Instead, they were
oriented normal to the ventricle and the ridge. This orientation
suggested that the cells migrated orthogonally into the
caudate-putamen--as migrating neural progenitors do from the
embryonic striatal neuroepithelium--not obliquely toward the tip of
the infusion cannula.
[0226] Two additional findings discounted the role of simple
chemoattraction in the migration of the ridge. First, the cells did
not begin to migrate as they were produced; they increased their
numbers along the ventricle for a period of more than a week, then
migrated en masse as a dense sheet of cells. Further, lesion of the
ipsilateral substantia nigra greatly increased the incidence of
migration. Both of these observations suggested a more complex set
of factors influencing the migration of the cells. These data did
not entirely rule out a role for chemoattraction in the migration
of the ridge, but they did indicate that a simple chemoattractive
effect could not by itself account for it.
[0227] Another mechanism that might have contributed to the radial
migration of neural progenitors in the adult striatum was the
reconstruction of the radial glial scaffold due to the neurotoxic
lesion, the mechanical injury done by the surgical implantation of
the infusion cannula, or both. Radial glia guide the migration of
neural progenitor cells in many regions of developing brain. They
are anchored along the ventricle and extend their processes
radially into the overlying parenchyma. They normally are
transformed into GFAP-IR astrocytes in the early postnatal period
once neuroblast migration is complete, and cease expression of
vimentin and nestin.
[0228] Freezing injury of the cortical plate in neonatal rats
inhibited radial glial transformation and caused the persistence of
glial expression of vimentin and nestin in the injured regions of
the adult brain (Rosen et al., Dev. Brain Res. 82:127-135, 1994).
Kainate lesion of the adult rat hippocampus induced radial glial
morphology and expression of nestin-IR and vimentin-IR in
astrocytes of the hippocampal subependymal zone, suggesting that
brain injury could stimulate a reversion of astrocytic phenotype to
one found in the embryonic brain (Clarke et al., Neuroreport
5:1885-1888, 1994).
[0229] The present immunohistochemical experiments do not provide
support for this phenomenon. Nestin-IR fibers were found in
abundance in radially-oriented fibers along the ventricle at
infusion day nine, but at later time points, they no longer
remained along the ventricle. As the cells of the ridge migrated
away from the subependymal region, so did the nestin-IR fibers.
Furthermore, immunostaining for vimentin, a specific marker for
radial glia, did not reveal any labeled fibers either in the ridge
or in the subependymal region. Thus, it is unlikely that any
astrocytes were reverted to their embryonic radial glial phenotypes
or that astrocytic reversion played a role in the radial migration
of the neural progenitor cells.
[0230] A newly-described mode of migration employed specifically by
neural progenitors in the adult mammalian brain elucidates the
tangential movement of these cells from the forebrain subependymal
zones to the olfactory bulbs (Lois et al., 1996, supra). Rostrally
migrating neuroblasts are densely packed and sheathed by GFAP-IR
astrocytes bordering their highly-restricted migratory pathway. The
migrating cells essentially form a solid stream of moving cells
within a tube of specialized glial guide cells. The neural
precursors in our experiments migrated as a sheet through the
striatal neuropil--not along a restricted path--and were not
associated with GFAP-IR cells. In fact, the proliferative
subependymal zone and the cellular ridge largely excluded GFAP-IR.
Thus, the mechanism of chain migration could not account for the
radial migration seen in the present studies.
[0231] Another mechanism possibly underlying the mass neural
progenitor migration was that cells of the striatum may have
altered their expression of growth factors, cell adhesion
molecules, or other substances in response to injury. In this
scenario, the striatum may have been stimulated to provide its own
chemoattractants or molecules that facilitate radial migration.
Alternatively, it may also have been induced to downregulate
expression of substances that inhibit migration.
[0232] Recent studies examining cell adhesion molecules in the
striatum and subependymal region provide particularly intriguing
insight. Highly polysialylated neural cell adhesion molecule
(PSA-N-CAM) immunoreactivity is intensely expressed in the
developing rodent striatum, but decreases as the animal matures
(Aaron and Chesselet, Neurosci. 28:701-710, 1989; Szele et al.,
Neurosci. 60:133-144, 1994). PSA-N-CAM expression, however,
persists along the adult forebrain subependymal region (Rousselot
et al., 1994; Szele et al., 1994, supra). Partial
decortication-induced striatal deafferentation dramatically
increased expression of PSA-N-CAM and another adhesion molecule,
L1, in the subependymal zone (Poltorak et al., J. Neurosci.
13:2217-2229, 1993; Szele and Chesselet, J. Comp. Neurol.
368:439-454, 1996). In the human brain, PSA-N-CAM expression is low
in normal striatum, but is increased in the striata of Huntington's
disease patients, particularly in the subependymal zone (Nihei and
Kowall, Ann. Neurol. 31:59-63, 1992).
[0233] Of special interest were the changes occurring in
fibronectin mRNA expression in the striatum after partial
unilateral frontal decortication (Popa-Wagner et al., Neuroreport
3:853-856, 1992). Fibronectin is one of a number of molecules that
have been shown to support neural migration in vitro (Fishman and
Hatten, J. Neurosci. 13:3485-3495, 1993). Fibronectin mRNA
hybridization was increased to a maximum level at 72 hours in the
portion of the striatum immediately under the wound cavity. This
early increase was interpreted as a component of the short-term
wound healing process. Fibronectin expression in the greater
ipsilateral striatum followed a longer-term increase, peaking at
about ten days post lesion. This secondary increase was interpreted
as the striatal response to deafferentation. Increases in
expression of two other mRNAs that code for N-CAM and alpha tubulin
followed only the early wound healing-related spatial and temporal
patterns.
[0234] The ten-day peak of striatal fibronectin mRNA expression
after deafferentation corresponds well to the delay of ridge
migration following striatal dopamine denervation in the present
studies. The delay of peak fibronectin mRNA expression may help
explain why the progenitor cells of the invention did not begin to
migrate radially until around the ninth day of infusion, and why,
when they finally did migrate, they migrated en masse. In animals
where the infusions and lesions were separated by several weeks,
the maximum lateral migration distances at two weeks of infusion
were dramatically reduced. This observation, too, is consistent
with the transient peak in striatal fibronectin expression. In the
few animals with ridges that did not also receive nigral lesions,
mechanical injury of the overlying cortex may have stimulated
enough fibronectin expression in the striatum to facilitate the
migration. Fibronectin, then, may be upregulated in response to
dopamine denervation of the ipsilateral striatum and, in turn, may
temporarily facilitate radial migration of neural progenitors from
the subependymal zone.
[0235] Another possible influence on radial migration of neural
progenitors in the adult striatum may stem from a secondary effect
by cell adhesion molecules. Infusion of N-CAM into the brains of
adult rats receiving stab wounds to various areas of the brain,
including striatum, inhibited astrocytic proliferation (Krushel et
al., Proc. Natl. Acad. Sci. USA 92:4323-4327, 1995). Astrocytes
release factors inhibiting neurite outgrowth and may thus inhibit
neural regenerative responses. Thus, denervation of the striatum,
and the associated increase in subependymal PSA-N-CAM may release
inhibition of neural regeneration.
[0236] The enhancement of the migration effect in
dopamine-denervated striatum may also have been related directly to
the loss of dopaminergic innervation. During embryonic development
of the striatum, immature neurons originate in the ventricular
region and migrate radially (Bayer, 1984, supra; Bayer and Altman,
Prog. Neurobiol. 29:57-106, 1987). The developmental migration of
striatal neurons takes place prior to dopamine innervation by
afferents from the midbrain. Stimulation of D.sub.2 dopamine
receptors on hypothalamic neurons dramatically attenuated
TGF.alpha. mRNA expression and pituitary growth (Borgundvaag et
al., Endocrinology 130:3453-3458, 1992). Although dopamine
receptor-mediated inhibition of TGF.alpha. expression has not been
studied in the subependymal zone, it is consistent with the
depressed incidence of migration in non-lesioned animals. Thus,
dopamine innervation during development may inhibit migration of
striatal cells as the forebrain dopaminergic innervation becomes
established.
[0237] Striatal dopamine may also contribute to the downregulation
of striatal TGF.alpha. early in postnatal development as
dopaminergic afferents become established. Dopamine denervation of
the adult striatum may mimic for striatal cells some of the local
chemical environmental cues normally only seen in the developing
striatum--for instance, a reduction of available ligand for
dopamine receptors expressed on striatal neurons. Striatal dopamine
innervation has also been linked in a reciprocal manner to
expression of extracellular matrix (ECM) molecules by astrocytes
(Gates et al., J. Chem. Neuroanat. 6:179-189, 1993). Interestingly,
TGF.alpha. is selectively elevated in the striata of PD patients
(Mogi et al., Neurosci. Lett. 180:147-150, 1994) similar to the
elevated expression in the embryonic striatum. If striatal
TGF.alpha. is regulated by dopamine innervation, this increase may
relate to the reduction of striatal dopamine and the consequent
release of inhibition of TGF.alpha. expression.
[0238] Whatever the underlying mechanism, the time course
experiment proved that subependymal cells could be stimulated to
increase their numbers and migrate radially en masse into the
adjacent striatum in the adult rat brain. Experiments in which the
location or the dose of TGF.alpha. infusion was varied showed that
movement of the striatal ridge and the gross numbers of cells
involved could be controlled. The characterization experiments
provided abundant evidence that the subependymal cellular expansion
and the dense striatal ridge were composed of neural progenitor
cells. The importance of these discoveries is discussed below,
together with their potential application for the treatment of
human neurodegenerative disease and traumatic brain injury.
[0239] The invention has now been explained with reference to
specific examples and embodiments. Other embodiments will be
suggested to those of ordinary skill in the appropriate art upon
review of the present specification.
[0240] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *