Notch-1 activation and dendritic atrophy
in prion disease
Nako Ishikura*†, Jared L. Clever*†, Essia Bouzamondo-Bernstein†, Erik Samayoa†, Stanley B. Prusiner‡§¶?,
Eric J. Huang†**††, and Stephen J. DeArmond†¶?††
Departments of *Pathology (Neuropathology),‡Neurology, and§Biochemistry and Biophysics, and¶Institute for Neurodegenerative Diseases, University of
California, San Francisco, CA 94143; and **Pathology Service, Veterans Administration Medical Center, San Francisco, CA 94121
Contributed by Stanley B. Prusiner, November 19, 2004
In addition to neuronal vacuolation and astrocytic hypertrophy,
dendritic atrophy is a prominent feature of prion disease. Because
increased Notch-1 expression and cleavage releasing its intracel-
lular domain (NICD) inhibit both dendrite growth and maturation,
Mountain Laboratory (RML) prions. The level of NICD was elevated
in the neocortex, whereas the level of ?-catenin, which stimulates
dendritic growth, was unchanged. During the incubation period,
levels of the disease-causing prion protein isoform, PrPSc, and NICD
increased concomitantly in the neocortex. Additionally, increased
levels of Notch-1 mRNA and translocation of NICD to the nucleus
correlated well with regressive dendritic changes. In scrapie-in-
fected neuroblastoma (ScN2a) cells, the level of NICD was elevated
compared with uninfected control (N2a) cells. Long neurofilament
protein-containing processes extended from the surface of N2a
cells, whereas ScN2a cells had substantially shorter processes.
Transfection of ScN2a cells with a Notch-1 small interfering RNA
decreased Notch-1 mRNA levels, diminished NICD concentrations,
and rescued the long process phenotype. These results suggest
that PrPScin neurons and in ScN2a cells activates Notch-1 cleavage,
resulting in atrophy of dendrites in the CNS and shrinkage of
processes on the surface of cultured cells. Whether diminishing
Notch-1 activation in vivo can prevent or even reverse neurode-
generation in prion disease remains to be established.
neurodegeneration ? scrapie ? Creutzfeldt–Jakob disease ?
conversion into nascent prions (1). Formation of prions involves
a conformational change in the precursor protein (2). In mam-
mals, the accumulation of prions is accompanied by neurode-
generation (3–6), whereas in fungi, the prion state is associated
with altered metabolism (7, 8). In both mammals (9, 10) and
fungi (11), fragments of the precursor proteins have been
refolded under cell-free conditions and shown to be infectious
upon introduction into the appropriate host.
In mammals, the prion diseases include Creutzfeldt–Jakob
disease of humans, scrapie of sheep, and bovine spongiform
in the CNS, resulting in presynaptic bouton degeneration, den-
dritic atrophy, vacuolation of neurons, and hypertrophy of
is formed from the precursor protein PrPCby a profound
conformational change (2). PrPCcontains three ?-helices (13),
at least one of which, along with some portion of the unstruc-
tured region, seems to refold into a ?-helix during the transfor-
mation to PrPSc(14, 15).
The tertiary structure of PrPScappears to encipher biological
information that defines a particular prion strain (10, 16–19).
Strains of prions differ phenotypically in their incubation times,
ability to infect animals of another species, and neuroanatomical
patterns of PrPScdeposition (5, 20, 21). When PrPScaccumulates
rions are infectious proteins that propagate by recruiting a
naturally occurring precursor protein and stimulating its
within neurons and their processes, neuropathological changes
that typify the degenerative process, including vacuolation of
synaptic regions and nerve cell death, are triggered. Quantitative
morphological, functional, neurochemical, and immunohisto-
chemical studies during the course of prion disease have iden-
tified a stereotypical progression of neurodegeneration (6, 12).
After initiation of disease in a group of neurons by intracerebral
inoculation of prions, disease spreads to other neurons and to
other brain regions by anterograde axonal transport of PrPScto
axon terminals. This is followed by presynaptic bouton degen-
eration and dendritic atrophy and, later, by nerve cell death.
To investigate the molecular events that underlie dendritic
atrophy, we studied the expression of genes that regulate den-
drite growth and maturation during CNS development. We
began by examining ?-catenin, which initiates dendritic growth
and maturation (22), and then measured Notch-1, which inhibits
both dendritic and axonal growth and maturation during neu-
ronal development and causes regression of mature dendrites
and axons (23–26). We report here that dendritic atrophy was
accompanied by increased levels of the Notch-1 intracellular
domain (NICD) in neuronal nuclei of prion-infected mice.
Elevated levels of NICD were also found in scrapie-infected
neuroblastoma (ScN2a) but not in uninfected control (N2a)
cells. Although long processes, also referred to as neurites,
extend from the surface of N2a cells, ScN2a cells exhibit much
shorter processes. To determine whether Notch-1 activation has
a role in the shortening of these processes, we transfected ScN2a
the normal long-neurite phenotype was rescued. These findings
suggest that Notch-1 activation may mediate dendritic atrophy in
the brains of humans and animals dying of prion disease.
Materials and Methods
Animals. All animal experiments were performed according to the
Guidelines of the Society of Neuroscience and the Institutional
Animal Review Committee of the University of California, San
Francisco. Animals were housed in pairs under diurnal lighting
conditions (12-h light–dark cycles). The macroenvironment was
controlled to provide a temperature of 20 ? 2°C and a relative
humidity of 45 ? 5%. Female CD1 mice were inoculated intrathal-
amically on the right with the RML strain of mouse prions, which
and terminal disease at ?150 dpi. They were killed by decapitation
at 30, 60, 90, 120, and 130–140 dpi. Age- and sex-matched controls
were inoculated in parallel with PBS.
Abbreviations: NICD, Notch-1 intracellular domain; PrPSc, disease-causing isoform of the
prion protein; ScN2a, scrapie-infected neuroblastoma cells; N2a, uninfected control cells;
siRNA, small interfering RNA; RML, Rocky Mountain Laboratory; dpi, days postinoculation;
NaPTA, sodium phosphotungstate.
†N.I. and J.L.C. contributed equally to this work.
?S.B.P. and S.J.D. have financial interests in InPro Biotechnology, Inc.
††To whom correspondence may be addressed. E-mail: email@example.com or
© 2005 by The National Academy of Sciences of the USA
January 18, 2005 ?
vol. 102 ?
Cell Culture. N2a and ScN2a cells were maintained in Eagle’s
minimum essential media with Earle’s salts containing 2 mM
L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium
pyruvate, 100 units of penicillin G per ml, 0.1 mg of streptomycin
sulfate?ml, and 10% FCS at 37°C in 5% CO2. To induce
differentiation, N2a and ScN2a cells were plated onto glass
coverslips that had been sequentially coated with 1 mg?ml
poly-L-lysine in 0.1 M borate buffer, pH 8.5, and 0.1 mg of mouse
laminin per ml in PBS, respectively. Alternatively, cells were
grown in 6-well or 10-cm dishes coated with polylysine?laminin
To stimulate neural process formation, cells were then cultured
in neurobasal medium containing the above-described antibiot-
ics, 2 mM L-glutamine, N2 supplement, and 10 ng?ml murine
2.5S nerve growth factor (Invitrogen).
PrPScin Synaptosomal Preparations. Synaptosomes were purified
from the neocortex as described (6). In brief, the neocortex was
dissected and pooled from two mouse brains. Three pools were
obtained at each time point for statistical analysis. All steps of the
isolation procedure were carried out quickly, and all solutions were
chilled on ice. The pools were suspended in 10 volumes (wt?vol) of
and homogenized in a Potter–Elvehjem glass homogenizer with 14
to 50-ml conical tubes and centrifuged at 1,500 ? g for 10 min in
a Beckman fixed rotor centrifuge to pellet cell debris and nuclei.
The supernatants were removed and the pellet resuspended for a
second slow-speed clarification process to obtain additional synap-
KA-30 centrifuge (Optima LE-8OK, Beckman Coulter) to obtain
a pellet enriched in synaptosomes. That pellet was resuspended in
10 volumes of RIPA lysis buffer (1 ? PBS, pH 7.4?1% igepal
CA-630?0.5% sodium deoxycholate?0.1% SDS), vortexed at 4°C
5 min to remove additional cellular debris from the synaptosomes.
For additional enrichment of PrPSc, the final synaptosomal
supernatants were subjected to two rounds of incubation with
sodium phosphotungstate (NaPTA) to selectively precipitate
PrPSc(18, 27, 28). Protein concentrations in the supernatants
were determined by bichromic acid assay. For the first precip-
itation, a volume of solution containing 2 mg of total protein was
obtained from each sample, and a stock solution of Sarkosyl was
added to a final concentration of 2% (wt?vol). These samples
were mixed with a stock solution containing 4% NaPTA and 170
mM MgCl2, pH 7.4, to obtain a final concentration of 0.32%
NaPTA. After 1-h incubation at 37°C on a rocking platform, the
samples were centrifuged for 30 min at 14,000 ? g in a micro-
centrifuge at room temperature (RT). For the second precipi-
tation, the pellets were resuspended in 0.25 ml of PBS with 0.1%
Sarkosyl containing 50 ?g?ml proteinase K (PK) to eliminate
PrPCand protease-sensitive PrPSc. After 1 h at 37°C on a rocking
platform, PK digestion was stopped by the addition of 2 mM
phenylmethylsulfonyl fluoride. To that solution, NaPTA was
added to a final concentration of 0.32% followed by incubation
at 37°C for 1 h on a rocking platform and then centrifugation at
14,000 ? g for 30 min at RT to precipitate protease-resistant
PrPSc. The pellets were resuspended in equal volumes of SDS
sample buffer and their proteins separated on 4–12% Bis-Tris
polyacrylamide gels and transferred to poly(vinylidene difluo-
ride) membranes for Western blot analysis recommended by the
manufacturer (NuPage gel system, Invitrogen). The gels were
loaded with aliquots containing equal amounts of protein based
on the protein measurements made just before the NaPTA
Western Blot Analysis of Neocortex and N2a?ScN2a Cells. Neocortex
dissected from mouse brains was homogenized in RIPA buffer.
For cultured cell lines, cells were scraped from plates, pelleted
at low speed, and lysed in 10 mM Tris?HCl?100 mM NaCl?10
mM EDTA?0.5% Triton X-100?0.5% deoxycholate, pH 7.5.
Protein concentrations were determined by using the bichromic
acid assay protein assay by using BSA as a standard (Pierce).
After SDS gel electrophoresis and transfer of proteins to poly-
(vinylidene difluoride) membranes, the blots were probed by
using the following antibodies: Notch-1 C-20 and ?-catenin E-5
from Santa Cruz Biotechnology (Santa Cruz, CA), cleaved
Notch-1 Val-1744 from Cell Signaling Technology (Beverly,
MA), GAPDH from Chemicon International (Temecula, CA),
and PrP-specific HuM-D13 (19). Proteins were detected with an
enhanced chemiluminescence system (Amersham Pharmacia).
Densitometry of autoradiograms was used to estimate the rel-
ative concentrations of target proteins.
Immunofluorescence Microscopy. Ten-micrometer-thick coronal
cryostat sections were mounted on glass slides and fixed in
methanol containing 1% hydrogen peroxide. For triple fluores-
(Santa Cruz Biotechnology) binding was detected by using
biotinylated horse anti-goat IgG (heavy and light chain) followed
by incubation with fluorescein-containing reagents of the tyra-
mide signal amplification Fluorescent System (PerkinElmer). To
detect neuron-specific mouse anti-NeuN antibodies (Chemi-
con), anti-mouse Alexa Fluor 568 (Molecular Probes) was used
as the secondary antibody. Nonspecific binding during this
mouse-on-mouse immunohistochemical step was blocked with
the Vector M.O.M. kit (Vector Laboratories). Vectashield
Mounting Media containing DAPI (Vector Laboratories) was
used to identify nuclei. N2a and ScN2a cells grown on coverslips
were fixed for 10 min with 4% formaldehyde in PBS and then in
1:1 methanol?acetone for 10 min before being stained for mouse
antineurofilament protein (NF200, NovoCastra, Newcastle,
UK), which was detected with anti-mouse Alexa Fluor 568.
Fluorescence images were recorded with Zeiss LSM 510 and
Leica TCS SP confocal microscopes.
Dendrite Length and Spine Density. Dendritic length and spine
density were measured as described (29). Golgi silver impregnation
was performed according to the instructions with the FD Rapid
GolgiStain Kit (FD NeuroTechnologies, Ellicott City, MD). Silver-
pyramidal neurons whose dendritic trees were relatively isolated
from other silver-impregnated neurons and that did not have
discontinuities or breaks in their dendritic trees were chosen for
quantification in each of three ill and three age-matched control
animals at several time points during the course of the disease. The
primary apical and total basal dendrite lengths of neurons in layer
4 of the somatosensory neocortex overlying the hippocampus were
measured at ?200. SCION IMAGE FOR WINDOWS, release ?4.0.2
(Scion, Frederick, MD) was the morphometric program used to
make the measurements of dendrite parameters.
Quantitative RT-PCR. Total RNA was isolated from normal and ill
extracting with chloroform. RNeasy columns (Qiagen, Valencia,
CA) were used to purify further the total RNA post-DNase-I
performed on total RNA with Taqman core RT reagents (Applied
Biosystems) by using random hexamers as primers. Amounts
ranging from 250 pg to 2 ?g of total RNA were used to create
standard curves, and 100 ng of experimental total RNA was used
PCR reaction. Taqman probes and primers were designed by using
PRIMER 3 software (Whitehead Institute Center for Genome Re-
Ishikura et al.
January 18, 2005 ?
vol. 102 ?
no. 3 ?
search, Cambridge, MA) and were purchased through Integrated
DNA Technologies (Coralville, IA). The PCR reactions were
carried out by using Taqman core PCR reagents (Applied Biosys-
tems) with 200 nM concentration of primers and 100 nM of
9700 sequence detector was programmed to an initial step at 50°C
for 2 min and by 95°C for 10 min, followed by 40 cycles of 95°C for
15 sec and 60°C for 1 min. Once the PCR was completed, each
sample was given a threshold cycle (CT) value, which is defined as
detection threshold. The analysis of the Taqman data was done by
using the comparative CTmethod (Applied Biosystems, Sequence
Detection System User Bulletin no. 2). In each case, GAPDH was
used as an endogenous reference.
RNA Interference. Three chemically synthesized siRNAs, Notch-
1-1 (nucleotides 473–491), Notch-1-2 (nucleotides 482–500), and
Notch-1-3 (nucleotides 1529–1547), against murine Notch-1
were purchased from Ambion. These siRNAs were transiently
transfected into N2a and ScN2a cells by using Lipofectamine
2000 per the manufacturer’s instructions (Invitrogen). An
siRNA targeted to GFP (GPF-22) was purchased from Qiagen
as a negative control in transfection experiments. Briefly, cells
were plated the day before transfection at the desired density in
maintenance media without antibiotics, typically 5,000 cells per
cm2. Cells were exposed to the siRNA-Lipofectamine mixture
for 5 h before the maintenance medium was replaced by differ-
entiation medium for the times indicated.
NICD Levels Increase During Prion Infection. Homogenates were
prepared from neocortex of prion-inoculated CD1 mice that
of ?-catenin and NICD. We observed a statistically significant 2-
to 3-fold increase in the level of NICD in prion-infected mice but
no change in ?-catenin (Fig. 1 a and b). Confocal microscopy
showed accumulation of NICD in the nuclei of many neocortical
neurons; in contrast, little or no NICD was located in the nuclei
of neurons in uninfected control cortex (Fig. 1c).
Increased NICD Levels Correlate with PrPScAccumulation. We found
in prion-infected mice. Because previous studies indicated that
the initial accumulation of PrPScin the neocortex is within
presynaptic boutons after its anterograde transport along
thalamocortical pathways (6), we measured PrPScconcentration
infection in CD1 mice. (a) Western blot analysis of NICD (Notch-1 C20 antibody),
?-catenin, and GAPDH in neocortical homogenates from four ill mice at 130 dpi
and four age-matched control mice. Identical results were obtained with the
cleaved Notch-1 Val-1744 antibody. (b) Densitometry estimates of the concen-
trations of NICD and ?-catenin relative to GAPDH show a statistically significant
2.5-fold increase in NICD concentration (Student’s t test;*, P ? 0.0001) but no
nuclei of layer IV neurons in RML-infected mice at 140 dpi. In uninfected control
neurons, merge shows small amounts of NICD mostly in the cytoplasm. A C-
to identify neurons. DAPI identifies nuclei.
Increased concentrations of NICD but not ?-catenin occur during RML
creased amounts of NICD, increased expression of Notch-1 mRNA, and regres-
sive changes in dendrites. (a) Kinetics of the log of neocortical PrPScaccumu-
lation in synaptosomes (filled squares) relative to NICD concentrations (filled
circles) during the course of prion disease. NICD levels in age-matched PBS-
inoculated mice are shown as controls (open circles). (b) A plot of synaptoso-
mal PrPScvs. NICD shows a high degree of correlation. (c) Quantitative RT-PCR
measurements of Notch-1 mRNA levels at three time points during the course
of prion disease (filled circles) relative to PBS-inoculated controls (open cir-
cles). (d and e) Camera lucida drawings of Golgi silver-stained dendritic trees
show one type of regressive change; compare PBS-inoculated age-matched
control cerebral cortex (d) with RML-infected cerebral cortex at 90 dpi (e).
Note that the Golgi method stains only a small percentage of neurons. (f and g)
Apical dendrite lengths (f) and numbers of apical dendrite branch points (g)
during RML infection (filled circles) compared with uninoculated age-
matched controls (open circles) are shown. Data points and bars represent
means and SEM, respectively, calculated from three independent experi-
ments.*, P ? 0.05;**, P ? 0.005;***, P ? 0.0001 by Student’s t test.
PrPScaccumulation in neocortical synaptosomes coincides with in-
www.pnas.org?cgi?doi?10.1073?pnas.0408612101Ishikura et al.
by using synaptosomal preparations. Further enrichment of
PrPScfrom synaptosomes was accomplished by selective precip-
itation with NaPTA (18). Compared with uninoculated controls,
the level of NICD in prion-inoculated mice increased progres-
sively throughout the incubation period, reaching statistically
significant increases at 120 and 130 dpi (Fig. 2a). The levels of
PrPScrose ?700-fold during the incubation period, whereas the
levels of NICD increased ?2.5-fold compared with controls. The
progressive increase of NICD concentration correlated well with
the accumulation of PrPSc(R ? 0.94) (Fig. 2b). A progressive
increase in neocortical Notch-1 mRNA expression, measured by
quantitative RT-PCR also followed accumulation of PrPSc(Fig.
2c). At 30 dpi, the levels of Notch-1 mRNA were similar in
control and prion-infected mice (n ? 3). By 90 dpi, a ?20%
increase in Notch-1 mRNA was detected, which rose to ?50%
(P ? 0.05) at 120 dpi. These in vivo results suggest a close
temporal relationship between PrPScaccumulation and elevated
levels of NICD and raise the possibility that PrPScdirectly or
indirectly triggers increased Notch-1 expression and cleavage.
PrPScAccumulation Results in Dendritic Atrophy.Lossandshortening
been reported (30), as has progressive loss of dendritic spines after
local accumulation of PrPScin mouse hippocampus (12). Here, we
examined 10 Golgi-silver-impregnated pyramidal neurons in layer
4 from each of three control (Fig. 2d) and prion-infected mice
(Fig. 2e) at four time points during the incubation period. Lengths
of apical and basal dendrites, the numbers of dendritic branch
points, and the numbers of dendritic spines were quantified as a
function of PrPScaccumulation in the neocortex. Only changes in
the lengths of apical dendrites (Fig. 2f) and the numbers of apical
dendrite branch points are shown (Fig. 2g), because all of the
regressive changes produced similar curves when plotted as a
function of the incubation period. These degenerative changes
as PrPScaccumulation continues to increase (Fig. 2a).
neurites compared with N2a cells. (a) Western blots show ?2? as much NICD in
ScN2a cells (cleaved Notch-1 Val-1744 antibody). GADPH was used to normalize
the data. (b and c) Phase-contrast microscopy shows that many N2a cells grew
neurites that are more than two cell diameters in length (b); in contrast, most
processes of ScN2a cells are less than two cell diameters in length (c). (d and e)
of both N2a (d) and ScN2a cells (e) are immunopositive for the high molecular-
weight neurofilament protein, NF200. N2a cells grew far more NF200-
in e applies to d.)
ScN2a cells have a higher concentration of NICD and fewer cells with
?30% in both N2a and ScN2a cells and rescues the long-neurite phenotype in
ScN2a cells. (a) NICD levels in N2a and ScN2a cells are reduced 3 d after transfec-
tion with 5 nM Notch-1 siRNA (Notch-1-1, Ambion); in contrast, 5 nM GFP siRNA
had no apparent effect. (b and c) Phase-contrast microscopy shows that ScN2a
with GFP siRNA (b) 3 d posttransfection and growth in differentiating medium.
expressed as a percentage of total cells. Data points and bars represent mean
For transfection experiments, ?1,000 cells were examined at each time point.
Open circles, control N2a cells; filled diamonds, ScN2a cells treated with 5 nM
filled triangles, untreated ScN2a cells.
An siRNA against Notch-1 decreases NICD concentration by a mean of
Ishikura et al.
January 18, 2005 ?
vol. 102 ?
no. 3 ?
Elevated NICD Levels Interfere with Growth of Neuritic Processes.
Similar to prion-infected brains, ScN2a cells had elevated levels of
NICD compared with N2a cells (Fig. 3a). Both the ScN2a and
control N2a cells were grown in a defined neurobasal medium
containing N2 supplement and 10 ng?ml nerve growth factor that
By convention, processes with a length of less than twice the cell
N2a cells grew numerous long neurites under these culture condi-
tions (Fig. 3b), whereas most ScN2a cells grew short processes (Fig.
3c). Immunohistochemistry shows that the cell bodies and neurites
of N2a and ScN2a cells contain high molecular-weight neurofila-
ment protein (NFP200), which also highlights the difference in
numbers of neurites (Fig. 3 d and e).
Inhibition of Notch-1 Activation Enables Normal Neurite Growth. To
test the possibility that the short-process phenotype is related to
elevated NICD levels, ScN2a cells were transfected with Notch-1
siRNA or GFP siRNA as a negative control. Three different
commercially available Notch-1 siRNAs were obtained and tested
for knockdown of gene expression in N2a and ScN2a cells. One of
N2a and ScN2a cells was used in all subsequent experiments.
Subconfluent cultures were transfected for 5 h with 5 nM or 50 nM
medium was replaced with the differentiating growth medium, and
the effects on NICD levels and neurite outgrowth were measured
over 72 h. Because both 5 and 50 nM Notch-1 siRNA produced
?40% decreases in Notch-1 mRNA by 48 h relative to untreated
cells, as measured by quantitative RT-PCR, all subsequent trans-
fections were performed by using 5 nM siRNA. In both N2a and
at 72 h (Fig. 4a), whereas 5 nM GFP siRNA had no detectable
effect. The levels of NICD were quantified by densitometry of
Western blots from three separate experiments.
At 72 h in differentiation medium, ?8% of nontransfected
ScN2a cells or ScN2a cells transfected with GFP siRNA had
surface neurites (Fig. 4 b and d); in contrast, ?27% of ScN2a
d). The number of N2a cells with neurites ranged from ?26% at
24 h (day 1) to ?40% at 72 h (Fig. 4d). The difference between
Notch-1 siRNA–transfected ScN2a cells and nontransfected N2a
cells was not statistically significant at 72 h (Fig. 4d). These
results argue that decreasing NICD concentration in ScN2a cells
by selectively knocking down Notch-1 mRNA expression re-
sulted in recovery of the normal neurite phenotype.
The results described above show a temporal association be-
tween PrPScaccumulation and increased levels of NICD in the
neocortex of mice infected with RML prions. Elevated levels of
NICD; as well as translocation of the NICD to the nucleus were
verified here, regressive dendritic changes were prominent in the
brains of these prion-infected mice (Fig. 2). Because dendritic
growth in the developing nervous system is inhibited by NICD,
our results raise the possibility that increased levels of NICD and
its translocation to the nucleus may be responsible for dendritic
atrophy in prion diseases. To explore this possibility, ScN2a cells
were studied to determine whether the relationship between
PrPScand Notch-1 in brains of prion-infected mice was recapit-
ulated in cultured cells. Compared with uninfected controls,
NICD was elevated ?50%, and the number of cells with long
neurites was significantly reduced in ScN2a cells.
To test the possibility that elevated NICD levels in ScN2a cells
are responsible for the shortened processes on the surface of
these cells, we used siRNA to diminish the level of NICD. Under
recovery of the normal neurite-length phenotype was observed.
These findings argue that the short-process phenotype in ScN2a
cells and, by inference, regressive dendritic changes in prion-
infected mice are due to increased levels of NICD.
It will be important to inhibit Notch-1 activation in mice
inoculated with prions and to determine whether the incubation
time is prolonged. Might mice continue to produce PrPScbut not
exhibit neurological dysfunction under such circumstances? Al-
though the results of the studies reported here show that
inhibition of Notch-1 activation restores dendritic processes on
the surface of cultured cells, it is unknown whether these events
occur in animals. If inhibition of Notch-1 activation does prevent
dendritic atrophy in vivo, it will be of interest to learn whether
neuronal vacuolation and astrocytic gliosis, which are generally
present in prion disease, are diminished concomitantly.
Because only the RML prion strain was used in both the mouse
and cultured cell studies described here, we must determine
whether Notch-1 activation occurs with other strains. Much evi-
dence indicates that different strains of prions reflect distinct
conformers of PrPSc(10, 16–19). It is also critical to establish
whether Notch-1 activation features in the pathogenesis of human
prion disease, including the inherited forms of these disorders.
The data presented here suggest that PrPScaccumulation in
plasma membranes (6, 31) directly or indirectly activates Notch-1
dendritic atrophy. It is notable that Notch-1 and the ?-amyloid
precursor protein (APP) are both cleaved by ?-secretase (32).
Cleavage of APP by ?-secretase is a critical step in the formation
of A?42 peptide that comprises amyloid plaques in Alzheimer’s
disease (AD). Additionally, elevated levels of Notch-1 protein (33,
34) and dendritic atrophy (35) are features of AD.
From the studies reported here, it is important to ask whether
inhibitors of Notch-1 expression or cleavage might be used as
pharmacotherapeutics for prion disease. Indeed, inhibitors of
?-secretase are being developed in an effort to find therapeutics
for AD. Whether inhibition of ?-secretase activity might also
delay cognitive decline secondary to synaptic degeneration in
Creutzfeldt–Jakob disease might be worth investigating.
We thank Peter Nelken and Amy Tang for additional technical support
and Hang Nguyen for editing the manuscript. This work was funded by
grants from the National Institutes of Health (AG10770, AG02132, and
AG021601). E.B.-B. was supported in part by the John Douglas French
Foundation for Alzheimer’s disease.
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