C: jag4 castillo.vp
J. Appl. Genet. 45(4), 2004, pp. 461-467
Tamoxifen and vitamin E treatments delay symptoms
in the mouse model of Niemann-Pick C
Eric C. BascuZan-Castillo1, Robert P. Erickson2, Christy M. Howison1, Robert J. Hunter2,Randall H. Heidenreich2, Chad Hicks3, Theodore P. Trouard4, Robert J. Gillies2,4
1 Department of Biochemistry and Molecular Biophysics, 2 Department of Pediatrics, 3 Optical Sciences Center, 4 BiomedicalEngineering Program, University of Arizona, Tucson, Arizona, USA
. Niemann-Pick C disease (NPC) is an irreversible neurodegenerative disorder without current treat-
ment. It is the result of deficient intracellular cholesterol movement. We investigated the effects of tamoxifen and
vitamin E (D-alpha tocopherol) treatment on patterns of weight loss and motor function in the mouse model
of Niemann-Pick C disease (Npc1-/-
mice). Tamoxifen has multiple metabolic effects, including reducing oxida-
tive damage, while vitamin E primarily has this property. Npc1-/-
mice were identified and treatment was initi-
ated at an approximate age of 21 days. Tamoxifen suspended in peanut oil was administered via intraperitoneal
injection (weekly, at a dose calculated to deliver 0.023 ìg/g/day). Vitamin E (25 IU) was administered orally via
gavage once a week. Weight loss and Rota-Rod performance were analyzed by using Kaplan-Meyer survival
curves. Tamoxifen treatment by itself significantly delayed weight loss (an endpoint of neurodegeneration)
in male and female mice compared to untreated controls. Motor function was evaluated by performance on
a Rota-Rod. Tamoxifen maintained Rota-Rod performance for about an extra week. Vitamin E treatment signifi-
cantly delayed weight loss in females only. Rota-Rod performance was maintained slightly longer in mice treated
with vitamin E. Simultaneous use of both treatments did not delay weight loss longer than tamoxifen-only treat-
ment but had a greater effect than either treatment alone on Rota-Rod performance and demonstrated a significant
positive effect on the early "learning curve" portion of the Rota-Rod evaluations. We found significant but rela-
tively small improvements in rate of disease progression by treating Npc1-/-
mice with tamoxifen and/or vitamin
E. Some sex differences in response and an early improvement in Rota-Rod performance suggest areas for further
: mice, neurodegeneration, Niemann-Pick C, Rota-Rod, tamoxifen, vitamin E.
protein was found to contain a sterol-sensingdomain consensus site and other motifs. This sug-
Niemann-Pick disease type C (NPC) is
gests a direct causative role for a mutant NPC1
a panethnic autosomal recessive disorder of un-
product in the altered cholesterol movement in
known pathogenesis (Vincent et al. 2003). A ma-
NPC. In spite of this likely role, the patho-
jor biochemical finding in this disorder is
physiological basis for the symptoms present in
the intracellular accumulation of unesterified cho-
NPC is unknown and gangliosides accumulate
lesterol within lysosomes and the Golgi apparatus.
as well (Zervas et al. 2001).
These findings have prompted the conclusion that
Previous studies with npc1
-/- mice revealed
NPC is a disorder of intracellular cholesterol traf-
a time-dependent accumulation of unesterified
ficking (Patterson et al. 1995). The NPC1
cholesterol in every organ except the brain. Subse-
recently been cloned in man (Carstea et al. 1997)
quently, however, it was found that the brain's ap-
and mice (Loftus et al. 1997), and the predicted
parent failure to accumulate cholesterol was due to
Received: May 17, 2004. Accepted: July 6, 2004.
Correspondence: R.P. Erickson, Department of Pediatrics/4341B., 1501 N. Campbell Avenue, P. O. Box 245073, Tucson, Ari-zona 85724-5073, e-mail: [email protected]
E.C. BascuZan-Castillo et al.
of heterozygous animals. Animals were kept at
non-neuronal loss due to demyelination (Dietschy
the University of Arizona Animal Care Facility
and Turley 2001). Treatment of NPC patients with
(PHS Assurance No. A-3248-01) on mouse chow
agents that lower somatic cholesterol has not had
containing 6% fat (or 10% for breeding mothers)
significant effects on the neurological symptoms
and water ad libitum
. At weaning (at about 21 days
(Patterson et al. 1993), although dimethyl
of age), tail tips were removed from mice
sulfoxide showed clinical improvement in one pa-
and DNA was prepared. Polymerase chain reac-
tient (Sakuragawa et al. 1988), and cholesty-
tions (PCRs) to identify genotypes at the Npc1
ramine and lovastatin had short-term benefits as
locus were performed using the primer pairs de-
assessed by magnetic resonance imaging (Sylvain
scribed in footnote 28 of Loftus et al. (1997).
et al. 1994). Nifedipine and probucol two agents
For PCRs we used 10 mmol/L Tris, pH 8.3,
that effectively reduce liver cholesterol did not al-
50 mmol/L KCl, 2.5 mmol/L Mg2+; 200 ìmol/L
ter the progression of CNS disease in npc1
dNTPs, 1.25U Taq
polymerase, and 1 ìmol
(Erickson et al. 2000). It is not certain whether
of each primer. DNA (20-40 ng) was added at
most lipid-lowering drugs successfully permeate
85°C, and cycles of 30 s at 95°C, 30 s at 61°C,
the blood-brain barrier, but a recent study found
1 min at 72°C ´ 35, and 10 min at 72°C were used.
The products were separated on 1.2% NuSieve
terol-mobilizing cyclodextrins decreased liver
cholesterol storage in npc1
-/- mice, but this route orintrathecal delivery had only slight effects on on-
set of neurological symptoms (Camargo et al.
Two different drugs were investigated as a treat-
In this study we analyzed the effects of
ment in disease progression. Tamoxifen (desic-
tamoxifen and vitamin E on the course of the dis-
cate, 99% concentrate, from Sigma-Aldrich, St.
ease in Npc1-/-
mice. Although tamoxifen is
Louis, MO.) was prepared by dissolving 16 mg
mostly known as an anti-estrogen used in the treat-
of the drug per 1 ml of peanut oil at room tempera-
ment and prevention of breast cancer, it has multi-
ture (23oC). Following dissolution, tamoxifen was
ple other effects relevant to NPC. Tamoxifen
filtered with the 0.8/0.2 ìm pre-filter, filter combi-
retards glycososphingolipid metabolism (Cabot
nation (Gelman, Suporfilter #4905) and trans-
et al. 1996) by inhibiting ceramide glycosylation
ferred to a sterile container. Peanut oil used as
(Lavie et al. 1997). It has lysosomotropic proper-
a control was simply filtered with a 0.8/0.2 ìm fil-
ties, altering vesicular transport recycling and se-
ter and transferred to a sterile container. D-alpha
cretory pathways (Altan et al. 1999), which could
tocopherol (brand "Natural Liquid Vitamin E"),
accentuate or ameliorate the NPC1 defect in cho-
amphiphiles mimics the NPC1
cellular phenotypeand endogenous amphiphiles could be dislodged
by tamoxifen (Lange and Steck 1998). It inhibitsglutamate-induced mitochondrial depolarization
mice were weaned, genotyped and
(Hoyt et al. 2000) and lowers serum cholesterol
separated according to sex when they were ap-
(Bilimoria et al. 1996). Orally administered
proximately 21 days old. Treatment was initiated
tamoxifen demonstrated a marked decrease in
at this time. The mice were divided into four
the development of lipid lesions in apolipoprotein
groups: control, tamoxifen treated, vitamin E
E knockout mice (Reckless et al. 1997). Finally, it
treated, and tamoxifen plus vitamin E treated.
can reduce oxidative damage (Custodio et al.
1994). Vitamin E was chosen as another
intraperitoneally with the drug suspended in pea-
nut oil on a weekly basis with a dosage delivering0.023 ìg/g/day (0.01 c.c./g. and assuming linearrelease). Mice receiving vitamin E were given
Material and methods
70 ìl (25 IU) of D-alpha tocopherol through oraladministration via a pipette on a weekly basis.
Control mice were either injected with plain pea-
NIH mutant mice from the BALB/cJ back-
nut oil (with a volume equal to tamoxifen treated
ground were maintained by brother-sister mating
mice) or were given no treatment.
Tamoxifen and vitamin E in NPC1 mice
mal weight; < 10 seconds for 3 trials forRota-Rod), the last time when the mouse crossed
All mice were evaluated using two criteria: weight
the threshold was used as the age of failure, i.e.
loss and Rota-Rod performance. Mouse body
a mouse might re-gain some weight or improve
weights were recorded on a Monday, Wednesday,
on the Rota-Rod for a measurement before again
Friday schedule. When the weight of any mouse
dropping below the threshold. In the case of
dropped to 80% of its maximum value, rapid dete-
a mouse's death before reaching the weight failure
rioration and disease progression were observed.
point, the day of death was considered the failure
This threshold was used as the weight failure
point. The median age of failure was then used as
point, when found in 2 consecutive measurements,
an estimate of the survivability of the group.
for Kaplan–Meyer analysis, and mice were
The chi-squares compare the Kaplan-Meyer prod-
euthanized shortly thereafter.
uct for each group.
a Rota-Rod test instrument (Ugo Basile, NY,USA). The Rota-Rod was driven at a constant rate
of 25 rpm. The mouse was given 3 trials onthe Rota-Rod, and the maximum trial time was re-
Control mice did not show any variation in disease
progression (according to the criteria measured),
the Rota-Rod for 10 seconds were considered to
comparing those treated with plain peanut oil ver-
sus those with no treatment. Consequently, no dis-tinction was made between the two groups.
Weights and Rota-Rod times for days on which
measurements were not taken were calculated via
A pilot study with tamoxifen injected subcutane-
linear interpolation. The data were then analyzed
ously showed a significant delay in the time
by using Kaplan-Meyer survivability plots, as well
of weight loss (data not shown). However, some
as intra-group averaging for the Rota-Rod data.
leakage along the needle track led to questions
The Kaplan-Meyer plots show the percentage
about precise dosage. Hence the current study with
of mice considered viable versus age. In the case
intraperitoneal injections was performed.
of fluctuation around a failure point (80% of maxi-
. Median weight failure points in days from Kaplan-Meyer survival plots
Vitamin E females
Tamoxifen & vitamin E
. Median Rota-Rod failure points in days from Kaplan-Meyer survival plots
Tamoxifen & vitamin E
E.C. BascuZan-Castillo et al.
a significant difference in disease progression,beating their control counterparts by 8 days(6072 days, 95% CI, Table 1). However, the com-bined treatment was perhaps slightly less effica-cious than tamoxifen alone.
The median Rota-Rod failure results are presentedin Table 2. Control mice showed no significant sexdifferences (58 days, 5661 days 95% CI). Amongtamoxifen-treated mice, there was a slight delaybefore failure on the Rota-Rod (64 days,5171 days 95% CI). Mice treated with vitamin Eshowed a 7-day improvement over controls, witha median failure age of 65 days (62–66 days,95% CI). Mice treated with vitamin E were notsignificantly different from mice treated withtamoxifen. Mice given both treatments, showedan 11-day increase over controls with a medianfailure age of 69 days (5874 days, 95% CI; Ta-ble 2). In addition, these mice showed a significantincrease in absolute Rota-Rod performance (timespent on rotating rod) in the early stages of diseaseprogression (Figure 1).
It is now clear that the neurodegeneration seen in
Figure 1. Average Rota-Rod performance for each group
NPC is an autonomous process in the central ner-
on an absolute scale: (A) male mice; (B) female mice
vous system. The impact of visceral pathology onthe neurodegeneration in NPC was studied by Lof-
The median weight failure points from
tus et al. (2002). They reintroduced the wild-type
Kaplan-Meyer plots are presented in Table 1.
gene into npc1
-/- mice by targeting its ex-
The experimental and control groups did not show
pression primarily to the CNS through the use of
any sex differences (except with vitamin E) in dis-
the prion protein promoter. Interestingly, neuro-
ease progression and are grouped together.
degeneration was prevented, life span was normal-
The control mice showed a median failure age of
ized, and the sterility of npc1
-/- mice was
59 days (57–61 days, 95% CI). Mice treated with
corrected. The rescue did not completely rectify
tamoxifen showed a significant improvement over
the accumulation of GM2 or GM3 gangliosides in
control mice. There was a 10-day improvement
some neurons and glia (Loftus et al. 2002).
over controls with a median failure age of 69 days
This observation is in agreement with other evi-
(52–79 days, 95% CI; Table 1). Female mice
dence that these higher order gangliosides are not
treated with vitamin E showed the most significant
obligatory players in NPC neurodegeneration (see
sex difference in disease progression. Male mice
below). Moreover, the persistence of visceral pa-
showed no improvement when treated with vita-
thology in the "rescued mice" reinforces the no-
min E, having a median failure age of 57 days
tion of an autonomous pathological process
(56–58 days, 95% CI). By contrast, females
occurring in the NPC brain. The cause of neuronal
death is unclear. As a means of identifying
the control mice by 11 days with a median failure
the mode of neuronal death in NPC, Erickson and
age of 70 days (67–74 days, 95% CI). In compari-
Bernard (2002) overexpressed Bcl2, an anti-
son to the tamoxifen group, the males in this group
apoptotic protein in transgenic mice, using
fared worse while the females fared about
the neuronal specific enolase promoter. Bcl2 pre-
the same. Mice given both treatments also showed
vents developmental programmed cell death, and
Tamoxifen and vitamin E in NPC1 mice
neuronal death caused by a variety of stimuli.
A re-investigation of these mice showed that
Cross breeding the neuronally-expressing Bcl2
unesterified cholesterol accumulation in the cere-
mice with npc1
-/- resulted in overexpression of
Bcl2 in npc1
-/- mouse brain neurons, but neuronal
subcortical regions was markedly decreased and
death was not spared (Erickson and Bernard
that a few mice lived longer than controls
2002). When the mice were treated with
(Gondre-Lewis et al. 2003). A pharmacological
minocycline, a tetracycline analog that crosses
approach was used to target another key synthetic
the blood-brain barrier, and reduces neuronal
enzyme higher upstream in the glycosphingolipid
death in ischemia and Huntington and Parkinson
synthetic pathway: glucosylceramide synthase.
diseases, neurodegeneration proceeded as in un-
Oral administration of the inhibitor of this en-
treated mice. This, and normal caspase1 levels in
zyme, N-butyldeoxynojirimycin, to npc1
-/- mouse brain, have suggested that neuronal
and cats resulted in reduced ganglioside accumu-
death in NPC does not proceed by a Bcl2
lation in the brain, accompanied by a modest delay
and minocycline-inhibitable apoptotic pathway.
in onset of neurological dysfunction and death
Thus, attempts to slow the progression
of the animals, and reduced Purkinje cell loss
of the disease with therapies targeted at other
(Zervas et al. 2001).
pathophysiological mechanisms seem warranted.
We found that tamoxifen treatment signifi-
As mentioned in the introduction, tamoxifen has
cantly delayed the characteristic loss of weight
a number of effects that could potentially amelio-
that occurs in Npc1-/-
mice (Table 1). This effect
rate the neurodegeneration of NPC. The multiple
was somewhat greater in males than in females
properties of tamoxifen may be relevant to its use
(which is not surprising given other sex differ-
in the current "cocktail" now in clinical trials for
ences in the symptoms of disease) (Erickson et al.
amyotrophic lateral sclerosis (Muscular Dystro-
2002) but not significantly so and our results are
phy Association – http://www.azstarnet.com/star/
from both sexes pooled. Tamoxifen by itself had
a smaller effect on Rota-Rod performance, which
The mode of action of tamoxifen in ameliorat-
assesses coordination versus ataxia. These results
ing the symptoms of Npc1-/-
is moot. The effects
could suggest that the characteristic loss of weight
of tamoxifen on ceramide glycosylation (Cabot
is not merely due to an inability to feed from
et al. 1996, Lavie et al. 1997) could still be rele-
the overhead water and pellet containers. In con-
vant to NPC. There is substantial accumulation of
trast to our finding of a slight benefit of
GM2, GM3, and other glycosphingolipids in the
intraperitoneal tamoxifen on neurodegeneration in
NPC brain, which has prompted studies of the role
mice, intracranial administration of
of these lipids in NPC neuropathogenesis.
tamoxifen to rat pups from 6 to 9 days of age inhib-
Taniguchi et al. (2001) found that GM1 accumu-
ited Purkinje dendritic outgrowth (Sakamoto et al.
lates primarily in neurons and astrocytes of npc1
2003). Thus, of these 2 drugs, vitamin E might be
mouse brain, whereas GM2 accumulates in neu-
more appropriate for further study.
rons and macrophages. GM1, which is normally
Vitamin E has long been known as an antioxi-
localized to synaptosomal membranes of neurons,
dant. Resent research has focused on particular
accumulates in the cytoplasm and other dendrites,
pathways that are affected by it, e.g. signaling
whereas GM2, which is typically absent from the
pathways (Rimbach et al. 2002) or superoxide pro-
neuronal soma, accumulates in perinuclear vesi-
duction (Ulker et al. 2003). We found no effect of
cles. In view of the postulated role of these
vitamin E in male mice, while female mice
showed a significant delay in weight loss. How-
dendritogenesis, these subcellular alterations
ever, sex differences were insignificant for
alone may contribute to neuronal dysfunction.
Rota-Rod performance so that data could be
A genetic approach aimed at reducing the lev-
pooled and vitamin E treated mice showed a mild
els of gangliosides by mating npc1
-/- mice with
improvement in this parameter.
Effects of tamoxifen plus vitamin E were gen-
the â-1-4GalNAc transferase gene responsible for
erally similar to those of tamoxifen or vitamin E
synthesis of GM2 and higher order gangliosides,
for both growth and motor performance. The com-
successfully reduced CNS accumulation of GM2
bined treatment was slightly better than either
and glycolipids GA1 and GA2, but did not im-
alone for Rota-Rod performance but not better for
prove the clinical phenotype or neuronal pathol-
weight maintenance. Perhaps it is the combined
ogy of the npc1
-/- mice (Liu et al. 2000).
E.C. BascuZan-Castillo et al.
anti-oxidant influence of these 2 drugs which is
Custódio JB, Dinis TC, Almeida LM, Madeira VM,
intramembranous inhibitors of lipid peroxidation.
When analyzing Rota-Rod average perfor-
Evidence for peroxyl radical scavenging activity.
mance (Figure 1) instead of Rota-Rod "time of
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Health Care for Women International, 23:98–118, 2002Copyright © 2002 Taylor & Francis0739-9332 /02 $12.00 + .00 CHILDBIRTH CARE-SEEKING BEHAVIOR IN CHIAPAS Linda M. Hunt, PhD Department of Anthropology and Julian Samora Research Institute, Michigan State University, East Lansing, Michigan, USA Namino Melissa Glantz, BA
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