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The Journal of Neuroscience, March 15, 2003, 23(6):1992
BRIEF COMMUNICATION
Neprilysin Gene Transfer Reduces Human Amyloid Pathology in
Transgenic Mice
Robert A.
Marr1,
Edward
Rockenstein2,
Atish
Mukherjee4,
Mark S.
Kindy5,
Louis B.
Hersh4,
Fred H.
Gage1,
Inder M.
Verma1, and
Eliezer
Masliah2, 3
1 Laboratory of Genetics, The Salk Institute for
Biological Studies, La Jolla, California 92037, Departments of
2 Neurosciences and 3 Pathology, The University
of California San Diego, La Jolla, California 92093, and
4 Department of Biochemistry, The University of Kentucky,
Lexington, Kentucky 40536, and 5 Department of Physiology
and Neuroscience, Medical University of South Carolina, Charleston, SC
29425
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ABSTRACT |
The degenerative process of Alzheimer's disease is linked to a
shift in the balance between amyloid- (A) production, clearance, and degradation. Neprilysin has recently been implicated as a major
extracellular A degrading enzyme in the brain. However, there has
been no direct demonstration that neprilysin antagonizes the deposition
of amyloid- in vivo. To address this issue, a lentiviral vector expressing human neprilysin (Lenti-Nep) was tested in
transgenic mouse models of amyloidosis. We show that unilateral
intracerebral injection of Lenti-Nep reduced amyloid- deposits by
half relative to the untreated side. Furthermore, Lenti-Nep ameliorated
neurodegenerative alterations in the frontal cortex and hippocampus of
these transgenic mice. These data further support a role for neprilysin
in regulating cerebral amyloid deposition and suggest that gene
transfer approaches might have potential for the development of
alternative therapies for Alzheimer's disease.
Key words:
neprilysin; Alzheimer's disease; lentivirus; gene
therapy; amyloid-; endopeptidase
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Introduction |
Abnormal production and accumulation
in the CNS of amyloid- (A) protein, a proteolytic product of
amyloid-precursor-protein (APP) metabolism, is currently being
investigated as one of the central mechanisms involved in the
pathogenesis of Alzheimer's disease (AD). Although in familial forms
of AD mutations in the APP and presenilin (PS) genes result in
increased production of A1-42, in sporadic
forms the mechanism is less clear. Some studies point to decreased
A1-42 clearance, and others suggest that a
shift in the balance between A production and degradation might be
responsible for the disease (Glabe, 2000 ).
Neprilysin (Nep) has been recently identified as a major extracellular
A degrading enzyme in the brain (Iwata et al., 2000). Neprilysin
(neutral endopeptidase, EC 3.4.24.11, enkephalinase, CD10) is a 97 kDa
cell surface-associated zinc metalloendopeptidase that functions to
degrade peptide signaling molecules in the circulatory, immune, and
nervous systems (Turner et al., 2001). Recent studies have shown that
neprilysin knock-out mice exhibit a gene dose-dependent increase in
A levels in the brain (Iwata et al., 2001) that is comparable with
that associated with presenilin mutations. Additionally, Nep is reduced
in areas vulnerable to plaque formation (Akiyama et al., 2001; Reilly,
2001; Yasojima et al., 2001a,b), and downregulation of neprilysin with
chemical inhibitors results in increased A concentrations in the
brain (Iwata et al., 2000). Infection of primary neurons with a Sindbis
vector expressing neprilysin also reduces A production in
vitro (Hama et al., 2001). Taken together, these studies suggest
that neprilysin plays a key role in the clearance of A. To further
investigate the role of neprilysin in amyloid deposition, a lentiviral
vector expressing human neprilysin (Lenti-Nep) was prepared and tested
in transgenic models of amyloidosis. This approach was favored over
other viral vectors because lentiviral vectors have been shown to
efficiently transduce cells of the CNS, and they are not cytotoxic
(Naldini et al., 1996; Dull et al., 1998; Miyoshi et al., 1998).
Furthermore, expression of the therapeutic product can theoretically be
sustained for the life span of the animal.
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Materials and Methods |
Cell culture and medium. 293T cells were cultured in
DMEM (Cellgro, Kansas City, MO) + 10% FBS (HyClone,
Logan, UT) + antibiotic/antimycotic (Life Technologies,
Grand Island, NY). The rat hippocampal neural progenitor (HCN) cell
line (Ray et al., 1995) was cultured in DMEM/Hams/F-12 (high
glucose) (Omega Scientific, Tarzana, CA) supplemented with
1 mM L-glutamine, (Sigma, St. Louis,
MO), 1× penicillin/streptomycin/fungizone (Life
Technologies), 1× N2 supplement (Life
Technologies), and 20 ng/ml basic fibroblast growth factor (bFGF) (Life Technologies) on plates coated with
poly-L-ornithine (Sigma) and laminin
(Fisher, Houston, TX). For differentiation, HCN cells were
grown in the above medium without bFGF, and in the presence of 0.5%
fetal bovine serum and 1 µM retinoic acid (Sigma).
Lentiviral vector production. Vector plasmids were
constructed for the production of third generation lentiviral vectors
that expressed the neprilysin gene. The human cytomegalovirus (CMV) promoter was used to drive expression of the transgenes (see Fig. 1a). Vector plasmids were also constructed expressing green
fluorescent protein (GFP) and a point mutant of neprilysin (E585V)
(Hama et al., 2001). Additional control experiments were performed by
heat inactivating (70°C for 20 min) the vector (Lenti-iNep). All
vectors were designed to be self-inactivating (Miyoshi et al., 1998)
and used the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) 3' to the transgene (Zufferey et al., 1999). The HIV-1
central poly-purine track was also located 5' to the promoter (Follenzi
et al., 2000). We produced lentiviral vectors using a four-plasmid
transfection system, as described previously (Dull et al., 1998;
Miyoshi et al., 1998). Briefly, 293T cells were transfected with vector
and packaging plasmids, the supernatants were collected, and vectors
were concentrated by centrifugation. The lentiviral vector titers were
estimated by measuring the amount of HIV p24 gag antigen with an ELISA
kit (Perkin-Elmer Life Science, Boston, MA) [100,000
transducing units (TU) per nanogram of p24] or by flow cytometry (below).
Flow cytometry. 293T cells were plated into 24-well plates
(coated with poly-L-lysine) (Sigma)
and counted before infection. Infections were done by making serial
dilutions of the lentiviral vector preparation in culture medium and
incubating overnight in a volume of 200 µl. The next day the
transduced cells were washed with fresh medium and incubated for 3-4
d. The transduced cells were then removed with an EDTA buffer and
washed in PBS. For Lenti-Nep and Lenti-NepX (Fig.
1a), the cell pellet was then resuspended in 50 µl
of PBS/antibody solution containing 10 µl of FITC-conjugated
anti-CD10 antibody (CD10F) (Research Diagnostics, Flanders, NJ) and incubated for 1 hr at 4°C. Afterward the cells were
washed twice in PBS and analyzed by flow cytometry. For Lenti-GFP, transduced cells were simply collected (as above) and directly analyzed
by flow cytometry. The percentage of cells positive was used to
determine the number of transducing units per unit volume.
Transgenic mouse generation and intracerebral injections of
lentiviral vectors. For this study two independent lines of human (h) APP tg mice were used. The first expressed mutant human amyloid precursor protein cDNA (hAPPV717F) under
the control of the platelet-derived growth factor promoter (PDAPP
minigene) (Masliah et al., 1996). The second expressed double-mutant
hAPP London (V717I) and Swedish (K670M/N671L) under the control of the
murine Thy1 regulatory sequences (Rockenstein et al., 2001). A total of
13 PD-hAPP tg mice from line J9M, aged 12-20 months, and 13 mThy1-hAPP
from line 41 (TASD41), aged 11-14 months, were injected with 3 µl of
the lentiviral preparations (1.5 × 107 TU) into the frontal cortex and
hippocampus (using a 5 µl Hamilton syringe, 0.25 µl/min). Mice
received unilateral injections (right side) to allow comparisons
against the contralateral side, with Lenti-Nep, Lenti-NepX (inactive),
Lenti-iNep (heat inactivated), Lenti-GFP, or vehicle alone. One month
after injection mice were killed, and the brains were
immersion-fixed in 4% paraformaldehyde for subsequent
immunohistochemical analysis.
Immunoblots. For the detection of neprilysin in
vitro, transduced cells (1.0 ng of p24 per cell) were lysed in
RIPA lysis buffer plus protease inhibitors (Complete mini)
(Roche, Indianapolis, IN). The lysates were analyzed by
SDS-PAGE on a 7% Tris-acetate polyacrylamide gel (NuPAGE)
(Invitrogen, Carlsbad, CA). Immunoblots were performed
with a primary anti-CD10 antibody (clone 56C6, 1:75 dilution,
overnight) (Research Diagnostics) and a secondary goat
anti-mouse-IgG-HRP antibody (1:5000, 2 hr) (Santa Cruz
Biotechnology, Santa Cruz, CA) and visualized by
chemiluminescence (Amersham Biosciences, Baied d`Urfe, Canada).
For the detection of neprilysin expressed in vivo, 3 µl of
Lenti-Nep (1.5 × 107 TU) was
injected directly into the hippocampus (using a stereotaxic frame). For
negative controls, mice were injected with Lenti-GFP at the same
concentration or with saline. One week later the brains were removed,
and the right hemispheres were homogenized in RIPA buffer plus protease
inhibitors (Complete mini) (Roche) on ice. Protein
concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA). The homogenates were
immunoprecipitated overnight (4°C) with 20 µl preswollen protein G
Sepharose (Amersham Biosciences) and 1 µl of anti-CD10
antibody (CLONE SN5c/L4-1A1, 1 mg/ml) (Ancell, Bayport,
MN). After washing, precipitated proteins were extracted in protein
loading buffer and analyzed by immunoblot as described above.
Neprilysin activity assay. Neprilysin activity was measured
as described previously (Li and Hersh, 1995). Briefly, whole cell or
tissue homogenates (293T or human kidney) were measured using glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide as a substrate. Protein
was quantified using the BCA reaction. The addition of phosphoramide (a
neprilysin inhibitor) caused a 95% reduction in activity.
Amyloid- degradation assays. Lentiviral
vectors were used to transduce 293T or HCN cells in a 24-well plate.
Two days after transduction the medium was removed, and the cells were
washed and incubated with medium spiked with
A1-42 or A1-40 (1000 pg/ml; Biosource International, Camarillo, CA) + 1 µM ZnCl2 (Sigma) (without supplements or serum). The medium was
removed 6-8 hr later and gently centrifuged to avoid cell
contamination. The medium was then assayed for
A1-42 or A1-40
using isoform-specific ELISA kits (Biosource
International). Cells infected with Lenti-GFP and Lenti-NepX
(E585V) were used as negative controls. Medium plus
ZnCl2 was used to determine background signal for the ELISA assays. Thiorphan (Sigma) was also used as a
negative control (150 µM).
Immunohistochemical and neuropathological analyses. The
fixed brains were serially sectioned at 40 µm with a Vibratome
2000 (Leica, New York, NY) and sections were immunolabeled
as described previously with antibodies against A (3D6) (Elan
Pharmaceutical, South San Francisco, CA), MAP2
(Chemicon, Pittsburgh, PA), or neprilysin (56C6)
(Research Diagnostics) followed by incubation with
FITC-conjugated secondary antibodies and were imaged with the laser
scanning confocal microscope (LSCM) (Mucke et al., 2000). The
percentage of the area of the neuropil occupied by A-immunoreactive (IR) plaques was used to estimate amyloid load (10 random fields from
three sections of the hippocampus), whereas the percentage of the area
of the neuropil occupied by MAP2-IR dendrites was used to evaluate the
damage or preservation of the neuronal structure. Additional
double-immunolabeling experiments were performed to ascertain
colocalization of lentiviral neprilysin expression and amyloid plaques.
For this purpose, vibratome sections were incubated with antibodies
against A (3D6) and Nep (56C6) (Research Diagnostics) and visualized with Tyramide Red (for Nep) and FITC (for A), followed by analysis with the LSCM as described previously (Rockenstein et al., 2001).
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Results |
Expression of active neprilysin from
Lenti-Nep-transduced cells
Human 293T cells were infected with a lentiviral vector expressing
wild-type human neprilysin (Fig.
1a). The transduced cells were
harvested and immunostained with an FITC-conjugated anti-neprilysin antibody, and subsequent flow cytometric analysis showed clear labeling
for neprilysin on the cell surface (Fig. 1b). The expression of neprilysin was also detected from transduced 293T and differentiated neural progenitor (HCN) cell lysates by immunoblot (Fig.
1c). Transduced cells were shown to express biologically
active neprilysin by a colorimetric activity assay (see Materials and
Methods). 293T cells infected with the Lenti-Nep vector [multiplicity
of infection (m.o.i.) 50] produced an activity of 617 nmol · min 1 · mg1,
whereas Lenti-GFP infected cells produced only 0.7 nmol · min1 · mg1.
Human kidney (highest neprilysin-expressing tissue) extract produced an
activity of 24 nmol · min1 · mg1.
Furthermore, 293T cells infected with serial dilutions of the Lenti-Nep
vector showed the ability to degrade synthetic
A1-42 in a dose-dependent manner (Fig.
1d), as measured by ELISA. Differentiated neural progenitor
cells (HCN) (Ray et al., 1995) infected with the Lenti-Nep vector
(m.o.i. 50) also showed the ability to degrade both
A1-42 (Fig. 1e) and
A1-40 (data not shown). This activity was
reduced by thiorphan, an inhibitor of neprilysin. Additionally, an
inactive mutant of neprilysin (NepX) was unable to degrade A (Fig.
1e). We conclude that Lenti-Nep can efficiently transduce
cells in vitro and generate biologically active
neprilysin.
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Figure 1.
a, Vector design showing the
neprilysin, inactive neprilysin (NepX), and green fluorescent protein
(GFP) expressing lentiviral constructs. The internal promoters driving
the transgenes are indicated by arrows (human
CMV). LTR sequences are shown on the ends (packaging signal,
 ). b, 293T cells transduced with Lenti-Nep (10 nl per well, or 0.015 pg p24 gag-antigen per cell) were immunostained
for neprilysin expression and analyzed by flow cytometry.
c, Immunoblot on 293T cells and differentiated rat
neural progenitors (HCN) transduced with the
Lenti-Nep or Lenti-GFP vector. d, 293T cells
transduced with serial dilutions of Lenti-Nep could degrade
A1-42 in a dose-dependent manner. Lenti-GFP transduced
cells were used as a negative control (GFP), whereas
medium alone was used to determine the baseline
(Medium). e, Differentiated neural
progenitors (HCN) infected with Lenti-Nep
(Nep; n = 2), Lenti-NepX
(NepX; n = 2) or Lenti-GFP
(GFP; n = 2) were used to degrade
A1-42 (as above). Values are presented as the
percentage of the average value from the GFP samples. Thiorphan was
also used as a negative control (Nep + Thior.;
n = 1).
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Neprilysin expression in the brain
Transgenic (APP-tg) and nontransgenic mice were injected with 3 µl of Lenti-Nep vector into the hippocampus. One week later we
removed the brains, and the injected hemispheres were homogenized in
RIPA buffer. Neprilysin was detected in the brain homogenates by
immunoblot (Fig. 2a). The
expression of neprilysin was also detectable by immunohistochemistry on
sections from injected mouse brains 1 month after injection (Fig.
2b). Expression was typically localized to the injection
site (covering a radius of >200 µm); however, in some cases human
neprilysin expression was detectable at more distant sites (data not
shown). The distribution of Nep expression was similar to the
distribution of GFP expression after the injection of a Lenti-GFP
vector (data not shown).
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Figure 2.
Expression of neprilysin in mouse brains.
a, Detection of human neprilysin expression by
immunoblot. Lenti-GFP and saline injections were used as negative
controls. The quantity of total protein extracted from mouse brains was
similar among all samples (data not shown). b, Detection
of human neprilysin by immunohistochemistry (Lenti-Nep, top
panels, arrows indicate examples of Nep-positive
cells; Lenti-GFP, bottom panels, APPtg,
J9M; Nontg, nontransgenic mouse; nep,
Lenti-Nep injected; vector, Lenti-GFP injected).
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Amyloid- deposits are reduced in
Lenti-Nep-transduced brains
To assess the potential anti-amyloidogenic effects of neprilysin
in vivo, a lentiviral vector expressing this gene was
injected into the CNS of APP transgenic mice from two independent lines that develop high levels of amyloid deposition. Amyloid plaques on the
Lenti-Nep-injected hemispheres were qualitatively smaller compared with
those on the contralateral side (Fig. 3,
compare A, C, D with B,
E, F). The use of double immunolabeling
for neprilysin and amyloid showed that the appearance of small plaques
colocalized with strong staining for neprilysin (Fig. 3, compare
G, H with I, J).
Amyloid quantification showed a significant decrease in the levels of
amyloid deposition compared with the contralateral site (57 ± 16%) (Fig. 4). In contrast, control
experiments in which transgenic mice were treated with Lenti-GFP,
Lenti-NepX (mutant Nep), Lenti-iNep (heat-inactivated Nep vector), or
saline alone showed comparable levels of amyloid deposition on both
sides of the brain (100 ± 16.5%). Similar results were also
obtained with Thioflavine-S staining (data not shown). Consistent with these findings, analysis of dendritic integrity with an antibody against MAP2 showed that treatment with Lenti-Nep was also associated with amelioration of the neurodegenerative process compared with the
contralateral side, as shown by an average 16.2 ± 7% increase in
MAP2 immunoreactivity on the ipsilateral side. In contrast, control-treated mice showed similar levels of neurodegeneration between
the two hemibrains, as shown by an average 3.7 ± 11% decrease in
MAP2 immunoreactivity on the ipsilateral side (p < 0.01).
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Figure 3.
Reduced amyloid plaque formation in mice injected
with Lenti-Nep. Shown are hippocampal sections from a representative
transgenic-APP mouse (TASD41) injected with Lenti-Nep into the
hippocampus and frontal cortex (right side injected: A,
C, D, G, H;
contralateral side: B, E,
F, I, J)
(amyloid = green). Double immunolabeling for
neprilysin and amyloid (G-J)
(nep = red).
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Figure 4.
Comparison of plaque burdens in Lenti-Nep and
control-treated transgenic mice. a, Mean absolute values
for amyloid staining in Lenti-Nep (Neprilysin) and
control-treated animals on the contralateral and ipsilateral
hemispheres (*p = 0.0007;
#p = 0.06; t test).
Error bars represent SE. b, Relative ratios of
ipsilateral (injected) versus contralateral (noninjected) plaque
burdens. Nep, Lenti-Nep injected; GFP,
Lenti-GFP injected; NepX, Lenti-NepX injected;
INep, heat inactivated Lenti-Nep injected;
Saline, saline injected. c, Scatter plot
showing individual and the average relative values of the neprilysin
(n = 12) and control (n = 14)
groups; p = 0.000001 (t test)
(TASD41: red squares, p = 0.01; J9M:
blue circles, p = 0.004;
t test). Error bars represent SD.
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Discussion |
The present study shows that Lenti-Nep reduced AD-like pathology
in transgenic mice. This finding is consistent with previous studies
showing that Nep plays an important role in the degradation of A in
the brain. For example, downregulation of neprilysin genetically or
with chemical inhibitors results in increased A concentrations in
the brain (Iwata et al., 2000, 2001), and gene transfer of Nep reduces
A concentrations in vitro (Hama et al., 2001).
Furthermore, it has been found that there is an inverse correlation
between the presence of neprilysin and susceptibility to amyloid plaque
formation in mice (Fukami et al., 2002). Similar findings have been
reported in humans; decreased neprilysin mRNA levels were found to be
associated with areas of high amyloid plaque burdens, colocalizing with
increased APP and PS mRNA levels (Yasojima et al., 2001a). This inverse
correlation is further supported by immunohistochemical localization of
neprilysin in similar areas of the human brain (Akiyama et al., 2001;
Yasojima et al., 2001b). Although it has been demonstrated that the
infusion of Nep inhibitors can accelerate A deposition and that Nep
induction is correlated with reduced A deposition (Iwata et al.,
2000; Mohajeri et al., 2002), to our knowledge no direct effect on the accumulation of A deposits had been established for Nep in
vivo. Our study demonstrated that Nep does have an inhibitory
effect on A deposition in mice, further supporting a regulatory role for this endopeptidase in AD. However, it is still unclear whether modulation of neprilysin expression or activity is intimately involved
in the etiology of AD, because genetic linkage analysis has shown no
correlation between the neprilysin locus and risk for development of AD
(Sodeyama et al., 2001; Oda et al., 2002).
The data presented for each mouse in Figure 4, b and
c, were controlled internally by individually comparing the
injected side with the contralateral side. This method of analysis
compensated for the variability in total plaque load seen between mice
(Fig. 4a) and demonstrated more clearly the localized
effects of neprilysin expression. The localization of these effects to
the injected hemisphere was expected, because wild-type neprilysin is a
type II integral membrane protein. Therefore, its effects were likely mediated through localized depletion of A near the sites of
expression. The mechanisms through which Nep might reduce amyloid
deposition include either increased degradation or reduced growth of
already existing plaques. Our studies support the possibility that both mechanisms are at work, because plaques found in regions strongly expressing neprilysin were observed to be smaller and more compact, and
in some cases, plaque load was reduced to <50% of that found on the
contralateral side (Figs. 3, 4). Considering the short time period
involved (1 month), it is unlikely that the greater load on the
contralateral side was solely the result of more rapid growth.
Therefore, at least in these more dramatic cases, it would seem that
plaques on the Lenti-Nep-injected sides were reduced.
Plaque assembly involves several stages that include A
oligomerization and profibral formation that might serve as the
nucleating nidus for A polymerization and fibril formation
(Zerovnik, 2002). The role of A oligomers and fibrils in
neurotoxicity in AD is still highly controversial; however, some
studies support a role for oligomers in neurotoxicity. It has been
suggested recently that oligomers of A are the major form
interfering with long-term potentiation in mice (Walsh et al., 2002).
The overexpression of neprilysin would likely reduce the local
concentration of A oligomers through the degradation of monomers and
possibly oligomers and fibrils directly. However, the potential
dissociation of fibrils could also increase the local oligomer
concentration and thus be harmful. Although our study did not directly
address this issue, it did demonstrate that neprilysin affects the A
dynamics, such that amyloid plaques either develop more slowly or
dissociate. Furthermore, this effect was associated with reduced
neurodegeneration, as revealed by MAP2 immunocytochemistry. This
apparent reduction in neurodegeneration could be the result of
protection of neurons from toxic A products, enhanced regeneration,
or simply upregulation of MAP2 expression. Regardless, this finding
implies that Nep gene transfer did not generate harmful products but
rather had a beneficial effect in vivo. In conclusion, our
study supports a role for neprilysin in the regulation of amyloid
deposition and highlights the potential use of gene therapy approaches
for treatment of Alzheimer's disease.
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FOOTNOTES |
Received Oct. 23, 2002; revised Dec. 4, 2002; accepted Dec. 27, 2002.
R.A.M. was supported in part by funds from the Canadian Institutes of
Health Research. M.S.K. is supported by Grant AG 19323 from National
Institute on Aging. This work was supported in part by funds from the
Alzheimer's Association (L.B.H.). F.H.G. is supported by National
Institutes of Health Grant AG08514, the Lookout Fund, the Fox
Foundation, and the National Parkinson Foundation. I.M.V. is an
American Cancer Society Professor of Molecular Biology and is supported
by grants from National Institutes of Health, the Wayne and Gladys
Valley Foundation, and the H. N. and Frances C. Berger Foundation.
E.M. is supported by National Institutes of Health Grants AG5131,
AG10689, and AG18440 and by a grant from the M. J. Fox Foundation
for Parkinson's Research. We thank Marylyn Gage for critical reading
of this manuscript.
Correspondence should be addressed to either of the following: Inder M. Verma, Laboratory of Genetics, The Salk Institute for Biological
Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, E-mail: verma{at}salk.edu; or Fred H. Gage, Laboratory of Genetics, The
Salk Institute for Biological Studies, 10010 North Torrey Pines Road,
La Jolla, California 92037, E-mail:
gage{at}salk.edu.
| |
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TOP
ABSTRACT
Introduction
