Journal of Neuropathology and Experimental Neurology
Copyright q 2000 by the American Association of Neuropathologists
Vol. 59, No. 12
December, 2000
pp. 1106 1117
Cholesterol Is Sequestered in the Brains of Mice with Niemann-Pick Type C Disease but
Turnover Is Increased
CHONGLUN XIE, MD, DENNIS K. BURNS, MD, STEPHEN D. TURLEY, PHD,
AND
JOHN M. DIETSCHY, MD
Abstract. In Niemann-Pick Type C (NPC) disease, the concentration of cholesterol increases with age in every tissue except
the brain. This study investigates whether accumulation of cholesterol might also occur within the cells of the central nervous
system (CNS), but be obscured by the simultaneous loss of sterol from myelin as neurodegeneration proceeds. At birth, when
there is little myelin in the CNS, the concentration of cholesterol is significantly elevated in every region of the brain in the
homozygous NPC mouse. At 7 wk of age, myelination is nearly complete. In the NPC mouse, however, there is striking
neurodegeneration and a reduction in both myelin protein and myelin cholesterol. Furthermore, net loss of cholesterol from
the CNS is much higher in the NPC mouse than in the control animal (2.23 versus 1.37 mg/day per kg) so that the concentration
of sterol in most regions of the brain is reduced. This neurodegeneration and loss of myelin cholesterol is not prevented by
deletion of either the low-density lipoprotein receptor or apolipoprotein E in the NPC animal. Thus, the cholesterol sequestration seen in every organ in NPC disease also occurs in cells of the CNS and may be etiologically related to the neurodegeneration.
Key Words:
ation.
Apolipoprotein E; Demyelination; Endosomes; Low-density lipoprotein receptor; Lysosomes; Neurodegener-
INTRODUCTION
Niemann-Pick Type C (NPC) disease in either humans
or mice is characterized by organomegaly at birth, liver
function abnormalities, and the development of progressive neurodegeneration with loss of myelin from some
regions of the central nervous system (CNS) (1–4). The
classical presentation of this disease in humans typically
involves a transient period of neonatal jaundice followed
by apparent normal development in early childhood.
However, behavioral abnormalities, ataxia, difficulties in
vertical gaze, and a variety of other neurological dysfunctions become manifest later in childhood, and these
signs of progressive neurodegeneration continuously
worsen until the child becomes physically and intellectually disabled. There is considerable variation in this
typical phenotype, however. Some children have much
more severe liver disease with marked hepatomegaly,
prolonged jaundice, ascites, and portal hypertension. Other individuals, by contrast, do not manifest either the hepatic or neurological symptoms until much later in life
(3, 4).
Recent studies have established that the mutations
causing this disease in both humans and mice affect a
protein designated NPC1 that is involved in the movement of cholesterol, and, possibly, other lipids, from late
From the Departments of Internal Medicine (CX, SDT, JMD) and
Pathology (DKB), The University of Texas Southwestern Medical Center, Dallas, Texas.
Correspondence to: John M. Dietschy, MD, Department of Internal
Medicine, The University of Texas Southwestern Medical Center, 5323
Harry Hines Blvd., Dallas, TX 75390-8887.
This research was supported by NIH Grant R37 HL09610 and by a
grant from the Moss Heart Fund.
endosomes and lysosomes to a pool of metabolically active sterol that can be used for the formation of other
products or for secretion from the cell (5–12). Thus, the
classical biochemical finding in the mouse homozygous
for this mutation is the time-dependent accumulation of
unesterified cholesterol in nearly every organ so that, by
7 wk of age, the whole-animal pool of sterol is increased
2.5-fold (;5,400 mg/kg body weight) above that found
in unaffected animals (;2,200 mg/kg) (13). Every organ,
except the CNS, participates in this sequestration of cholesterol although the relative increase in sterol concentration varies from ;10-fold in liver to only ;1.3-fold in
tissues like striated muscle (13). The exception to this
observation is the CNS where there is a significant reduction in the pool of unesterified cholesterol in the homozygous NPC mouse (253 mg/kg) compared to control
animals (305 mg/kg) (13). A similar reduction in the concentration of cholesterol in the brains of humans with this
disease has also been described (3, 14).
The cellular pathway responsible for this sequestration
of unesterified cholesterol is now fairly well understood.
NPC1 apparently is involved in the intracellular movement of sterol that enters cells through the clathrin-coated-pit pathway (6, 10, 11, 15). Low-density lipoprotein
receptors (LDLR) clustered in these coated pits bind lipoproteins containing either of 2 ligands: apolipoprotein
B-100 (apoB-100), that is contained in circulating lowdensity lipoproteins (LDL), and apolipoprotein E (apoE),
that directs chylomicron remnants (CMr) to the liver (16–
19). These particles are endocytosed, the vesicles are
acidified, and an acid hydrolase hydrolyzes sterol esters
to unesterified cholesterol (20, 21). In some manner that
is still poorly understood, NPC1 is responsible for moving this unesterified cholesterol to other metabolically active areas of the cell for further processing or secretion
1106
NEURAL CHOLESTEROL ACCUMULATION IN NPC DISEASE
(11, 22–26). The pattern of cholesterol sequestration in
different organs, therefore, reflects the importance of each
of these tissues in the clearance of cholesterol carried in
LDL (LDL-C) and CMr (CMr-C) from the plasma (13).
However, most of the net movement of cholesterol
through the tissues of the body does not utilize this coated-pit pathway. The majority of such sterol is synthesized
de novo in the extrahepatic organs, transported to the
plasma membrane of the cells of these tissues, and then
transferred to high-density lipoproteins (HDL-C) and esterified by the enzyme lecithin:cholesterol acyltransferase
(27–29). This cholesteryl ester is selectively taken up by
the endocrine tissues and liver using a class B, type I
scavenger receptor (SR-BI) and then either metabolized
to steroid hormones or excreted as biliary cholesterol and
bile acids (30–33). In contrast to the coated-pit pathway,
this movement of newly synthesized cholesterol to the
plasma membrane and through the HDL/SR-BI pathway
appears to be fully functional in NPC disease and may
explain why development of the fetus and newborn takes
place essentially normally in this disease (34). Presumably, abnormalities in organs like the liver and spleen
become clinically evident only as cholesterol begins to
cycle through the coated-pit pathway late in fetal development and during the neonatal period.
As neurodegeneration is such a key feature of the clinical syndrome of NPC disease, it is important to elucidate
whether these same transport processes also regulate cholesterol balance across the cells of the CNS and whether
this same sequestration of cholesterol takes place in neurons when NPC1 is mutated. Several studies have demonstrated that all, or nearly all, of the cholesterol necessary for development of the brain and spinal cord comes
from synthesis in situ (35–40). There is currently no evidence for the movement of LDL-C or HDL-C from the
plasma into the CNS (41). Other studies, however, suggest that during nerve growth and repair the axon may
utilize an extracellular source of lipoprotein cholesterol
(42). While the CNS does not contain apoB-100, several
different cell types synthesize apoE, and neurons express
LDLR as well as other members of the LDL receptor
gene family (43–45). The possibility exists, therefore,
that during neuron growth and remodeling there is movement of cholesterol into these cells through a coated-pit
pathway, and such sterol may become sequestered in the
cells of the CNS in NPC disease. However, proving this
possibility is difficult since the CNS is the only organ in
the body that normally has 2 different pools of unesterified cholesterol, i.e. the sterol present in the plasma
membranes of the neurons and support cells (3–4 mg/g)
and the sterol packed into the myelin sheaths formed by
oligodendrocytes (20–40 mg/g). If cholesterol does accumulate in neurons causing cell death with loss of myelin, then the net effect might be a decrease in the overall
1107
concentration of cholesterol in the CNS. Thus, these studies were undertaken in the homozygous NPC mouse for
several reasons: 1) to determine if cholesterol does, in
fact, accumulate in the cells of the CNS; 2) to establish
whether the fall in the concentration of sterol in the whole
brain with aging is due to the loss of myelin-associated
cholesterol; and 3) to examine whether deletion of LDLR
activity or apoE prevents the neurodegeneration and loss
of myelin sterol typical of NPC disease.
MATERIALS AND METHODS
Animals and Diet
Heterozygous NPC mice (NPC1/2) with a BALB/c background were bred with each other or with homozygous LDLR
knockout (LDLR2/2) or homozygous apoE knockout (apoE2/2)
animals (15, 46). From these crosses, 4 groups of mice were
ultimately derived and these included mice that were control
animals (NPC1/1) or were homozygous for loss of NPC1 function (NPC2/2). In addition, mice were generated that were homozygous for deletion of both NPC1 and LDLR activity (NPC2/
2
/LDLR2/2) or NPC1 activity and apoE (NPC2/2/apoE2/2). The
genotypes of the NPC and LDLR animals were identified using
PCR protocols, and the genotype of the apoE animals was determined by Western blot analysis (8, 13, 46, 47). After weaning, these animals were housed in groups in plastic colony cages in rooms with alternating 12-h periods of light and dark.
Since no gender differences exist in the parameters studied in
these experiments, the experimental groups contained equal
numbers of males and females. All animals were fed ad libitum
a low-cholesterol, pelleted basal rodent diet (No. 7001, Harlan
Teklad, Madison, WI) that contained 0.016% (wt/wt) cholesterol and 5% (wt/wt) total lipid. All studies were carried out in
the fed state during the final hour of the dark phase of the light
cycle. In most experiments, animals were studied at 7 wk of
age. In 1 experiment, cholesterol concentrations were measured
in both 1-day-old and 7-wk-old mice. In another study, wholebrain cholesterol concentrations were determined in animals at
ages varying from 1 day to 10 wk. All experimental protocols
were approved by the Institutional Animal Care and Research
Advisory Committee.
Tissue Cholesterol Concentration
Animals were exsanguinated from the inferior vena cava and
the major tissues were removed, weighed, and saponified (40).
The remaining carcass, composed mainly of skeleton, muscle,
and adipose tissue, was also saponified. The cholesterol concentration in these tissue samples was measured by gas-liquid
chromatography and expressed as mg of cholesterol per g wet
weight of tissue (mg/g) (48). For measurement of the total cholesterol pool in brain and the whole animal, these values were
expressed as the mg of cholesterol present per kg body weight
(mg/kg). It should be noted that these data were derived from
the peak on gas-liquid chromatography that co-chromatographed with authentic cholesterol. In extracts from the CNS
of newborn mice, a small additional peak corresponding to desmosterol was also usually observed. Thus, in the Results section, the concentration of both cholesterol and desmosterol is
given for these newborn animals.
J Neuropathol Exp Neurol, Vol 59, December, 2000
1108
XIE ET AL
Cholesterol Synthesis In Vivo
Each animal was injected intraperitoneally with 50 mCi of
[3H]water. One hour later, the animals were anesthetized and
exsanguinated, and the major tissues were removed and saponified. After isolation of the tissue sterols and quantitation of
their 3H content, the rates of cholesterol synthesis in each tissue
were determined and expressed as the nmol of [3H]water incorporated into sterols per hour per g (nmol/hr per g) (49–51).
The rate of incorporation of [3H]water into sterols by the tissues
was converted to an equivalent mg quantity of cholesterol assuming that 0.69 3H atoms were incorporated into the sterol
molecule per carbon atom entering the biosynthetic pathway as
acetyl CoA (52). From such data, the rate of cholesterol synthesis in these mice each day was calculated and these rates
were then normalized to a constant body weight of 1 kg (mg/
day per kg).
Tissue Dissection and Morphology
Animals were killed, and brains and spinal cords were removed. In the case of specimens used for biochemical studies,
brainstems and cerebella were amputated from the cerebral
hemispheres and sectioned in the fresh state in the axial plane.
Unfixed cerebral hemispheres were sectioned in the coronal
plane. Anatomic landmarks were identified under a dissecting
microscope and fresh tissue from appropriate regions, including
cerebral cortex, midbrain, cerebellum, brainstem, and spinal
cord, was isolated for cholesterol assay.
For morphological studies, fresh brains were immediately
placed into 10% neutral, buffered formalin and fixed at room
temperature for a minimum of 48 h. Following fixation, the
cerebrum from each animal was sectioned in the coronal plane,
and the brainstem, cerebellum, and spinal cord in the axial
plane. Fixed sections were dehydrated sequentially in graded
alcohols and xylene, paraffin embedded, and sectioned for histological study at 4 mm. Serial paraffin sections were rehydrated, stained with hematoxylin and eosin (H&E) and with luxol
fast blue/PAS/hematoxylin, and evaluated by light microscopy
for the presence of neuronal storage material and other structural alterations.
Myelin Protein and Cholesterol Concentration
Brain myelin was isolated by sucrose density gradient centrifugation as described (53). Brains were weighed and homogenized in 0.32 M sucrose (20 ml/g) and layered over an equal
volume of 0.85 M sucrose. After centrifuging at 75,000 3 g
for 30 min, myelin was collected from the interface. Aliquots
of this fraction were saponified, and the cholesterol concentration was determined by gas–liquid chromatography. Myelin
protein concentration was also assayed (54). These data were
converted to mg of cholesterol or protein per g wet weight of
brain (mg/g).
Calculations
The data from all of these experiments are presented as mean
values 6 1 SEM. The Student unpaired t-test was used to compare the various sets of data for significant differences at the p
, 0.05 level.
J Neuropathol Exp Neurol, Vol 59, December, 2000
Fig. 1. Cholesterol concentration in various tissues of
NPC1/1 and NPC2/2 mice. Panels (A) and (B), respectively,
show the concentrations in 8 different tissues in NPC1/1 and
NPC2/2 animals at 7 wk of age. Panels (C) and (D) show the
same measurements in animals 1 day of age. Each group includes nearly equal numbers of males and females. The measurements represent mg of cholesterol per g wet weight of each
organ. Each value represents the mean 6 1 SEM for 6–10 animals in each group. Asterisks identify those values in the
NPC2/2 mice that are significantly different (p , 0.05) from
those in control NPC1/1 mice of the same age.
RESULTS
The phenotypic findings in the NPC mouse are illustrated in Figure 1. In control, NPC1/1 animals at 7 wk of
age, the concentration of cholesterol in every tissue, except the CNS and several endocrine glands (not shown),
varies from approximately 1.5 to 5 mg/g (A). Nearly all
of this sterol is unesterified and is largely found in the
plasma membrane of the cells of these tissues. The average concentration of cholesterol in the whole brain is
3–4-fold higher, and this sterol is all unesterified and
found in both cell plasma membranes and myelin. In
NPC2/2 animals the concentration of unesterified cholesterol is significantly elevated in nearly every organ, although this increase varies from only 1.3-fold in muscle
to 10-fold in liver (B). This sterol is localized in both
plasma membranes and intracytoplasmic vesicles. Only
in the whole brain is the mean cholesterol concentration
significantly lower in the NPC2/2 mice (13.6 6 0.1 mg/
g) compared to the control animals (15.0 6 0.1 mg/g).
In these 7-wk-old NPC2/2 mice there are also striking
abnormalities in the neurons of every region of the CNS.
As is evident in Figure 2, vacuolated cytoplasmic storage
material is present in virtually all areas of the CNS, including neurons in the cerebral cortex (E), cerebellum
(F), brainstem (G), and spinal cord (H).
NEURAL CHOLESTEROL ACCUMULATION IN NPC DISEASE
As shown in Figure 3, the concentration of cholesterol
in the cerebrum and cerebellum of the NPC1/1 mice
equals about 12 mg/g, but these values rise to almost 30
mg/g in the more heavily myelinated areas of the midbrain, brainstem, and spinal cord (A). As is also apparent,
there are significant decreases in the concentration of
cholesterol in several of these regions in the NPC2/2 animals and, importantly, the magnitude of this decrease is
proportional to the concentration of cholesterol in that
particular region, i.e. the greatest loss of sterol is seen in
those regions that have the highest concentrations of
myelin-associated cholesterol. Most of these regions also
have lower rates of cholesterol synthesis (B).
That the CNS of these 7-wk-old mice has less myelin
is further supported by the data in Table 1. There is a
significantly lower concentration of myelin protein (6.8
versus 10.5 mg/g) (A) and myelin cholesterol (3.1 versus
4.9 mg/g) (B) in the NPC2/2 animals. However, the ratio
of cholesterol-to-protein in the remaining myelin is unchanged (C). Taken together, these various results confirm that the lower concentration of cholesterol in the
CNS at 7 wk is largely accounted for by less myelinassociated cholesterol and this may have obscured the
sequestration of sterol in the cells of the CNS in NPC
disease.
In order to investigate this possibility, however, it was
necessary to make measurements under circumstances
where there is virtually no myelin in the CNS. As illustrated in Figure 4, the brain of the mouse has a mean
concentration of cholesterol equal to only 3.4 6 0.7 mg/
g at 1 day of age. Myelination occurs rapidly over the
next 3 wk, however, and at the end of this time the concentration of sterol in the whole brain exceeds 12 mg/g.
For purposes of comparison, this profile of myelination
is very different from that seen in an ungulate animal like
the sheep where the laying down of myelin cholesterol
occurs largely in the last half of gestation so that the
newborn is more fully developed and mobile at birth.
Even though relatively little LDL-C has cycled through
the coated-pit pathway at 1 day of age, the concentration
of cholesterol in every organ of the NPC2/2 pups is still
significantly elevated above that seen in the NPC1/1 control animals, as is also shown in Figure 1C, D. Most
importantly, however, at this early stage of development
where there is virtually no myelination, the concentration
of cholesterol in the whole brain is now also significantly
elevated (3.82 6 0.06 versus 3.45 6 0.07 mg/g) as is the
concentration of desmosterol (1.07 6 0.03 versus 0.90 6
0.02 mg/g). Furthermore, the magnitude of this elevation
is similar to that seen in most of the other organs (D).
Not only is this true for the whole brain but as shown in
Figure 5, the concentration of sterol is elevated in every
region of the CNS. It should be noted that in the NPC1/1
animals, the concentration of cholesterol in these regions
in the 1-day-old mice varies between only 3 and 5 mg/
1109
g, values that are well below those seen in the 7-wk-old
animals (Fig. 3). Thus, in the near absence of myelin, the
same level of cholesterol elevation seen in the other organs (Fig. 1D) is also seen in all regions of the CNS.
Although these findings establish that cholesterol does
accumulate in the CNS in NPC disease, the fact that neurodegeneration and demyelination are occurring simultaneously implies that sterol turnover in the brain must
be accelerated in this disorder. Studies were next undertaken to measure this turnover and to compare it to the
rate of cholesterol movement through the other organs of
the whole animal. As summarized in Table 2, the turnover
of cholesterol acquired from the diet and de novo synthesis is significantly higher in the NPC2/2 mice (177 mg/
day per kg) than in control animals (128 mg/day per kg)
(B). However, since the whole-animal sterol pool is so
much larger in the mutant mice (5,460 mg/kg) (A), this
daily rate of turnover accounts for only 3.1% of the
whole-animal pool in the NPC mice while the control
NPC1/1 animals turn over 5.5% of their pool each day
(C). Between 6 and 8 wk of age, the brains of the control
NPC1/1 mice accumulate an average of 0.79 mg/day per
kg of sterol (F) and synthesize 2.16 mg/day per kg (G).
As a consequence, these animals must be losing an average of 1.37 mg of cholesterol per day per kg from the
brain (H). During this same time, however, there is actually a net loss of 0.44 mg/day per kg from the sterol
pool of the brain of the NPC mice (F) and a significantly
lower rate of sterol synthesis (G). As a consequence, the
brain of these mutant animals lose significantly more cholesterol each day (2.23 mg/day per kg, H). Thus, the
NPC2/2 mouse is turning over a greater percentage
(0.67% per day) of the pool of cholesterol present in cell
membranes and myelin of the brain than is the NPC1/1
mouse (0.42% per day) (I).
The final experiment explored whether this accumulation of cholesterol in neurons, and the subsequent cell
death and apparent loss of myelin, could be ameliorated
by interfering with the putative uptake of sterol through
a coated-pit pathway in these cells. As the LDLR and
apoE are present in the CNS, and as this receptor and
ligand are very important in other organs for uptake of
cholesterol through the coated-pit pathway, the effect of
deleting each of these proteins in the NPC2/2 mouse on
the histological, biochemical, and survival aspects of
these animals was investigated. As shown in Figure 6,
there is little difference in the profile of lipoprotein-cholesterol in the NPC1/1 (A) and NPC2/2 (B) animals, although the mutant mice have a slightly elevated concentration of cholesterol carried in both LDL and HDL, as
has been reported previously (13, 15). The NPC2/2/
LDLR2/2 mice, however, have a markedly elevated concentration of LDL-C (C), while the NPC2/2/apoE2/2 animals have markedly elevated concentrations of
cholesterol carried in very low-density lipoprotein
J Neuropathol Exp Neurol, Vol 59, December, 2000
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XIE ET AL
1111
NEURAL CHOLESTEROL ACCUMULATION IN NPC DISEASE
TABLE 1
Myelin Protein and Cholesterol Concentration in the
Whole Brain of 7-Week-Old NPC1/1 and NPC2/2 Mice
NPC1/1
A. Myelin protein
concentration
(mg/g)
B. Myelin cholesterol
concentration
(mg/g)
C. Myelin
cholesterol/protein ratio
(mg/mg)
1/1
NPC2/2
10.5 6 1.3
6.8 6 0.6*
4.9 6 0.9
3.1 6 0.4*
0.47 6 0.09
0.45 6 0.05
2/2
Both the NPC
and NPC
mice used in these measurements were 7 weeks of age. Myelin protein and cholesterol
concentrations were assayed in the myelin fraction after preparative ultracentrifugation of whole brain homogenates. Each
value represents the mean 6 1 SEM for 8–10 animals. The
asterisks identify those values in the NPC2/2 animals that are
significantly different (p , 0.05) from those in the control
NPC1/1 mice.
Fig. 3. Cholesterol concentration (A) and rate of cholesterol synthesis (B) in 5 major areas of the CNS in NPC1/1 and
NPC2/2 mice at 7 wk of age. Each value represents the mean
6 1 SEM for 8–12 animals in each group. The asterisks identify those values in the NPC2/2 animals that are significantly
different (p , 0.05) from those in control NPC1/1 mice.
(VLDL) and intermediate density lipoprotein (D), as
would be anticipated from previous work. Loss of function of LDLR or apoE has significant and predictable
effects on the rate of cholesterol uptake through the coated-pit pathway and the accumulation of unesterified cholesterol in the cells of all organs outside of the CNS in
the NPC mouse (15). However, this is not true inside the
CNS. As shown by the examples in Figure 7, there are
no detectable differences in the morphology of neurons
in any region examined. The brains of the NPC2/2/
LDLR2/2 (C) and NPC2/2/apoE2/2 (D) animals are indistinguishable from those of the NPC2/2 (B) mice in terms
of the presence of cytoplasmic storage material and loss
of neurons. Similarly, as illustrated in Figure 8, the decrease in myelin-associated cholesterol in the midbrain,
brainstem, and spinal cord seen in the NPC2/2 mice is
not prevented when either LDLR activity or apoE is deleted from the animals. Finally, the gross neurological
abnormalities that occur in the NPC mice are unaltered
in the other 2 groups and lifespan is not improved in the
NPC2/2/LDLR2/2 (78 6 0.8 days, 111 animals) or NPC2/2/
apoE2/2 (85 6 1.6 days, 72 animals) animals compared
to that seen in the NPC2/2 (79 6 1.0, 57 animals) mice.
Thus, while there is little doubt that in NPC disease, unesterified cholesterol accumulates in the cells of the CNS,
as in all other tissues of the body, this sequestration, and
the accompanying neurodegeneration and apparent loss
of myelin, is not ameliorated by deletion of either the
LDLR or apoE.
DISCUSSION
These studies provide the first direct measurements in
support of the concept that in NPC disease there is cholesterol accumulation in the cells of the CNS, just as there
is in the cells of all other organs of the body (Figs. 1, 5)
(13, 15). This accumulation of sterol and other lipids presumably accounts for the perinuclear vesicular inclusions
seen in neurons in virtually all regions of the brain and
spinal cord (Fig. 2), and is consistent with the observation
that these inclusions fluoresce strongly under ultraviolet
←
Fig. 2. Histological sections of multiple CNS regions from NPC1/1 (A–D) and NPC2/2 (E–H) mice obtained at 7 wk of age.
Sections include parasagittal frontal cerebral cortex (A, E), cerebellar cortex (B, F), basal pontine neurons from brainstem (C,
G), and lumbar spinal lower motor neurons (D, H). Neurons of NPC2/2 animals from all levels of the neuraxis contain storage
material, visible as prominent vacuoles within neuronal perikarya (arrows). Sections of the cerebellar cortex also reveal significant
loss of Purkinje cells in the NPC2/2 animals (F) compared to NPC1/1 mice (B); residual degenerating Purkinje cells in the NPC2/2
animals (arrow) contain cytoplasmic vacuoles similar to those encountered in other neurons in NPC2/2 mice. H&E, 31,000.
J Neuropathol Exp Neurol, Vol 59, December, 2000
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XIE ET AL
Fig. 4. Cholesterol concentration in the whole brain of the
mouse and sheep at different times during fetal and neonatal
development. The gestation period for the mouse and sheep is
approximately 3 wk and 21 weeks, respectively. The average
cholesterol concentration in the whole brain of these 2 species
was determined before and after birth as indicated in the diagram. Each value represents the mean 6 1 SEM for 5–8 fetuses
or newborn animals at each time point. The data for the sheep
have been partially published previously (38, 41).
light when these tissues are treated with filipin (55). Unlike what happens in other organs, however, this accumulation is apparently associated with death of neurons,
loss of myelin, and excretion of excessive amounts of
cholesterol from the CNS (Table 2). As a consequence,
while the cholesterol concentration in virtually every other organ in the body increases with age in this disease
(Fig. 1), the overall concentration of sterol in the CNS
actually decreases (Fig. 3) (13). Nevertheless, these data
imply that the same abnormality in intracellular cholesterol transport found in all other organs of the NPC
mouse also exists in the cells of the CNS, a conclusion
that is further supported by recent studies in isolated neurons from this animal model (56).
Fig. 5. Cholesterol concentration in 5 major areas of the
brain in NPC1/1 and NPC2/2 animals at 1 day of age. These
pups had access to their mother’s milk for approximately 24 h
before these measurements were made. Each value represents
the mean 6 1 SEM for 6–8 animals in each group. The asterisk
identifies those values in the NPC2/2 animals that are significantly different (p , 0.05) from those in control NPC1/1 mice.
In these other organs, sequestration of unesterified cholesterol is dependent upon the uptake of lipoproteins that
contain either apoB-100 or apoE and that utilize the
LDLR present in coated pits. A second receptor, LDL
related protein (LRP), may also be important as a backup
transporter for apoE-containing lipoproteins, particularly
when LDLR activity is abrogated (16–18). The magnitude of lipoprotein cholesterol uptake through these
mechanisms and the rate of unesterified cholesterol sequestration can be altered by manipulating the function
of these ligands and receptors. For example, increasing
the amount of cholesterol flowing from the intestine to
the liver in apoE-containing CMr increases the rate of
TABLE 2
Net Cholesterol Turnover in the Whole Animal and Brain of 7-Week-Old NPC1/1 and NPC2/2 Mice
NPC1/1
A.
B.
C.
D.
E.
F.
G.
H.
I.
Whole animal cholesterol pool (mg/kg)
Whole animal cholesterol turnover (mg/day per kg)
Whole animal cholesterol turnover rate (% of pool per day)
Whole brain cholesterol pool at 6 weeks (mg/ kg)
Whole brain cholesterol pool at 8 weeks (mg/ kg)
Whole brain cholesterol accumulation rate (mg/day per kg)
Whole brain cholesterol synthesis at 7 weeks (mg/day per kg)
Whole brain cholesterol turnover (mg/day per kg)
Whole brain cholesterol turnover rate (% of pool per day)
2,174 6 46
128 6 5
5.5
322 6 5
333 6 11
0.79
2.16 6 0.12
1.37
0.42
NPC2/2
5,460 6 148*
177 6 10*
3.1
335 6 18
328 6 23
20.44
1.79 6 0.13*
2.23
0.67
Except for the values of the brain cholesterol pools in mice 6 and 8 weeks of age, all measurements were carried out in NPC1/1
and NPC2/2 at 7 weeks of age. The cholesterol pools, synthesis rates, and turnover rates are normalized to a constant wholebody weight of 1 kg and represent mean values 6 1 SEM for determinations in 10–15 mice in each group. The asterisks identify
those values in the NPC2/2 animals that are significantly different (p , 0.05) from those in the control NPC1/1 mice.
J Neuropathol Exp Neurol, Vol 59, December, 2000
NEURAL CHOLESTEROL ACCUMULATION IN NPC DISEASE
Fig. 6. Distribution of the plasma cholesterol in major lipoprotein fractions of NPC1/1 (A), NPC2/2 (B), NPC2/2/LDLR2/2
(C), and NPC2/2/apoE2/2 (D) mice. All animals used in this experiment were 7 wk of age. Plasma was pooled from 5–6 animals
in each group and the lipoproteins in this plasma were separated
by fast protein liquid chromatography. Note that the scale for
cholesterol content is very different in the different panels.
accumulation of sterol in the liver but not in the other
organs of the NPC mice (13). This effect is seen in the
present study even in the 1-day-old NPC pups that had
access to cholesterol-rich breast milk for 24 h and, consequently, manifested an exaggerated elevation of hepatic
unesterified cholesterol (Fig. 1D). Deletion of apoE in
such animals interferes with this accumulation. Similarly,
the rate of cholesterol sequestration in the extrahepatic
organs is proportional to the level of LDL-C uptake in
these tissues, and this pattern of accumulation can again
be altered when LDLR activity is abrogated (15). However, such sequestration apparently is associated with relatively little cell death, at least up to 7 wk of age, so that
the pool of cholesterol in the cells, and in the whole animal, simply increases as a function of age. Thus, the
homozygous NPC mouse essentially establishes a new,
quasi-steady state where each day about 80 mg of cholesterol per kg body weight is taken up into the tissues
through the coated-pit pathway and the pool of unesterified sterol in the whole animal expands by approximately
70 mg/day per kg (34). Under these conditions, some
organs such as the liver begin to manifest functional abnormalities, but most organs apparently function well
even though their stores of unesterified cholesterol are
expanded.
These experiments indicate, however, that the situation
is quite different in the cells of the CNS. Many studies
suggest that growth and repair of nerve cells, and their
myelination, require a source of cholesterol associated
1113
with apoE and receptors such as LDLR and LRP (57–
60). Thus, while most of the sterol required for development and differentiation of the CNS may come from
de novo synthesis, there may also be important internal
recycling of cholesterol within the CNS to neurons that
requires lipoprotein receptors and takes place through a
coated-pit pathway. Presumably, cholesterol taken up
through this pathway would become sequestered in the
neurons (Fig. 2), as in all organs outside the CNS, in
NPC diseases (Fig. 1). However, unlike the cells of these
other organs that apparently tolerate this stored cholesterol relatively well, neurons in various regions of the
CNS begin to die, and there is loss of myelin-associated
cholesterol (Table 1). This leads to a near doubling of the
rate of net cholesterol excretion from the CNS (Table 2),
so that the average concentration of sterol in the brains
of these mice (and humans) with NPC disease actually
decreases (Fig. 3) (3, 14).
Only recently has the mechanism of this cholesterol
excretion been elucidated. Brain expresses a unique cholesterol 24-hydroxylase that apparently converts excess
sterol in the CNS to 24S-hydroxycholesterol (61–63).
This oxysterol can penetrate the blood-brain barrier and
appears in the venous outflow from the brain. Utilizing
several different methods, it is now possible to calculate
this rate of cholesterol excretion in 3 different species.
By measuring the net appearance of 24S-hydroxycholesterol in the venous drainage of the brain, the rate of net
sterol excretion from the CNS in humans weighing about
70 kg equals ;0.09 mg/day per kg (0.02% per day of the
pool of cholesterol in the CNS) (64). Using an isotope
exchange method in baboons weighing about 23 kg, this
flux rate is found to equal ;0.18 mg/day per kg (0.04%
per day of the pool) (65). Direct measurement of the rate
of cholesterol excretion from the CNS in mice weighing
only 0.021 kg yields a value of ;1.37 mg/day per kg
(0.42% per day of the pool) (Table 2). Thus, the rate of
cholesterol excretion from the CNS is a function of animal weight. It should be noted that the rate of sterol turnover in the whole mouse (128 mg/day per kg, Table 2)
is about 15-fold greater than turnover in the whole human
(8–10 mg/day per kg), as is the turnover of cholesterol
in the CNS of this species.
One of the important implications to come from these
observations is that such measurements in the experimental animal or human may provide a quantitative measure of the rate of neurodegeneration taking place in a
particular pathological state. For example, in humans
with Alzheimer disease, the plasma concentration of 24Shydroxycholesterol is elevated 25% and 40%, respectively, above the level found in normal subjects or patients
with depression (66). In NPC disease in the mouse, where
the neurodegeneration is more florid (Fig. 2), cholesterol
excretion from the brain is elevated 63%. If these observations are confirmed in other pathological states, the fact
J Neuropathol Exp Neurol, Vol 59, December, 2000
1114
XIE ET AL
Fig. 7. Composite figure demonstrating the histological appearance of lumbar spinal motor neurons from NPC1/1 mice (A)
compared to NPC2/2 mice (B), and to NPC2/2/LDLR2/2 (C) and NPC2/2/apoE2/2 (D) animals. Prominent vacuoles are visible
within the neuronal cytoplasm of all NPC2/2 mice (arrows). The absence of either LDLR (C) or apoE (D) had no effect on either
the appearance or the distribution of neuronal storage material. H&E, 31,000.
that cholesterol released from dying cells is excreted from
the CNS may provide a means for assessing the magnitude of nerve cell death in other neurodegenerative disorders or after trauma.
However, these studies do not provide a measure of
the rate of cholesterol recycling among cells within the
CNS. The fact that the level of sterol sequestration in the
brain and spinal cord of the NPC mice (Fig. 5) is as great
as in various organs outside the neuraxis suggests that
this recycling is quantitatively substantial. While it is reasonable to assume that this process involves apoE and
LDLR, deletion of each of these proteins does not prevent
sterol sequestration in the neurons and neurodegeneration
(Figs. 7, 8). The LDLR is important in organs outside
the CNS for clearing plasma lipoproteins containing either apoB-100 or apoE. However, abrogation of LDLR
activity leads to accumulation of only LDL-C in plasma
since LRP readily takes over the function of clearing
apoE-containing remnant lipoproteins (Fig. 6C). Presumably, therefore, LRP or one of the other members of this
receptor family are active within the CNS (67). Failure
of deletion of apoE to interfere with this recycling process, however, is less easy to explain since a number of
J Neuropathol Exp Neurol, Vol 59, December, 2000
previous experiments indicate that this ligand is important for nerve repair or remodeling (58–60). Nevertheless, while an important role for apoE has also been suggested during the repair of peripheral nerves, such repair
apparently also occurs normally in animals lacking apoE
(68). Thus, there may be redundancy at this level of the
recycling process, both in the peripheral and central nervous systems, that allows some other apolipoprotein to
function as a ligand for the uptake process.
In summary, these studies have established that the defect in intracellular cholesterol transport manifest in every other organ also exists in the CNS when NPC1 is
mutated. Unlike these other tissues, however, this sequestration of sterol and other lipids leads to nerve cell
death, demyelination, and excessive cholesterol excretion
from the brain and spinal cord. Deletion of either the
LDLR or apoE alone does not alter this process. Clearly,
there are at least 2 areas where future studies should be
focused that may yield new information on the role of
cholesterol movement across and within the CNS in neurodegeneration in NPC disease. First, the experimental
means are now available for manipulating the rate of net
cholesterol movement across the brain and spinal cord.
NEURAL CHOLESTEROL ACCUMULATION IN NPC DISEASE
Fig. 8. Cholesterol concentrations in 5 major areas of the
CNS in NPC1/1, NPC2/2, NPC2/2/LDLR2/2, and NPC2/2/apoE2/2
mice at 7 wk of age. Each value represents the mean 6 1 SEM
for 6–8 animals in each group. The asterisk identifies those values in the NPC2/2, NPC2/2/LDLR2/2, and NPC2/2/apoE2/2 mice
that are significantly different from those in control NPC1/1 animals. The values in the NPC2/2/LDLR2/2 and NPC2/2/apoE2/2
mice are not significantly different from those in the NPC2/2
animals.
Second, by developing NPC mice that lack more than 1
lipoprotein ligand or receptor, it may be possible to also
manipulate the rate of cholesterol recycling within the
CNS. Such studies may yield valuable information on the
role of cholesterol in neurodegeneration not only in NPC
disease, but also in other degenerative diseases as well.
ACKNOWLEDGMENTS
The authors wish to acknowledge Jeff Graven, Brian Jefferson, and
Elizabeth Moore for excellent technical assistance and Merikay Presley
for preparation of the manuscript. The original colony of NPC mice in
Dallas was established from animals generously supplied by Dr. Peter
G. Pentchev of the National Institute of Neurological Disorders and
Stroke.
REFERENCES
1. Morris MD, Bhuvaneswaran C, Shio H, Fowler S. Lysosome lipid
storage disorder in NCTR-BALB/c mice. I. Description of the disease and genetics. Am J Pathol 1982;108:140–49
2. Higashi Y, Murayama S, Pentchev PG, Suzuki K. Cerebellar degeneration in the Niemann-Pick type C mouse. Acta Neuropathol
1993;85:175–84
3. Pentchev PG, Vanier MT, Suzuki K, Patterson MC. Niemann-Pick
disease type C: A cellular cholesterol lipidosis. In: Scriver CR,
Beaudet AL, Sly WS, et al, eds. The metabolic and molecular
bases of inherited diseases. 7th ed. New York: McGraw-Hill,
1995:2625–39
4. Jan MMS, Camfield PR. Nova Scotia Niemann-Pick disease (type
D): Clinical study of 20 cases. J Child Neurol 1998;13:75–78
5. Pentchev PG, Boothe AD, Kruth HS, et al. A genetic storage disorder in BALB/C mice with a metabolic block in esterification of
exogenous cholesterol. J Biol Chem 1984;259:5784–91
1115
6. Blanchette-Mackie EJ, Dwyer NK, Amende LM, et al. Type-C Niemann-Pick disease: Low density lipoprotein uptake is associated
with premature cholesterol accumulation in the Golgi complex and
excessive cholesterol storage in lysosomes. Proc Natl Acad Sci
USA 1988;85:8022–26
7. Sokol J, Blanchette-Mackie EJ, Kruth HS, et al. Type C NiemannPick Disease. Lysosomal accumulation and defective intracellular
mobilization of low density lipoprotein cholesterol. J Biol Chem
1988;263:3411–17
8. Loftus SK, Morris JA, Carstea ED, et al. Murine model of Niemann-Pick C disease: Mutation in a cholesterol homeostasis gene.
Science 1997;277:232–35
9. Carstea ED, Morris JA, Coleman KG, et al. Niemann-Pick C1 disease gene: Homology to mediators of cholesterol homeostasis. Science 1997;277:228–31
10. Liscum L, Klansek JJ. Niemann-Pick disease type C. Curr Opin
Lipidol 1998;9:131–35
11. Neufeld EB, Wastney M, Patel S, et al. The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo. J Biol Chem 1999;274:9627–35
12. Kobayashi T, Beuchat M-H, Lindsay M, et al. Late endosomal
membranes rich in lysobisphosphatidic acid regulate cholesterol
transport. Nature Cell Biol 1999;1:113–18
13. Xie C, Turley SD, Pentchev PG, Dietschy JM. Cholesterol balance
and metabolism in mice with loss of function of Niemann-Pick C
protein. Am J Physiol 1999;276:E336–E344
14. Vanier MT. Lipid changes in Niemann-Pick disease Type C brain:
Personal experience and review of the literature. Neurochem Res
1999;24:481–89
15. Xie C, Turley SD, Dietschy JM. Cholesterol accumulation in tissues
of the Niemann-Pick type C mouse is determined by the rate of
lipoprotein-cholesterol uptake through the coated-pit pathway in
each organ. Proc Natl Acad Sci USA 1999;96:11992–97
16. Ishibashi S, Herz J, Maeda N, et al. The two-receptor model of
lipoprotein clearance: Tests of the hypothesis in ‘‘knockout’’ mice
lacking the low density lipoprotein receptor, apolipoprotein E, or
both proteins. Proc Natl Acad Sci USA 1994;91:4431–35
17. Ishibashi S, Perrey S, Chen Z, et al. Role of the low density lipoprotein (LDL) receptor pathway in the metabolism of chylomicron
remnants.A quantitative study in knockout mice lacking the LDL
receptor apolipoprotein E, or both. J Biol Chem 1996;37:22422–27
18. Rohlmann A, Gotthardt M, Hammer RE, Herz J. Inducible inactiviation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants. J Clin
Invest 1998;101:689–95
19. Borén J, Lee I, Zhu W, et al. Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the
modulation of its binding activity by the carboxyl terminus in familial defective apo-B100. J Clin Invest 1998;101:1084–93
20. Goldstein JL, Dana SE, Faust JR, et al. Role of lysosomal acid
lipase in the metabolism of plasma low density lipoprotein. J Biol
Chem 1975;250:8487–95
21. Brown MS, Anderson RGW, Goldstein JL. Recycling receptors:
The round-trip itinerary of migrant membrane proteins. Cell 1983;
32:663–67
22. Liscum L, Dahl NK. Intracellular cholesterol transport. J Lipid Res
1992;33:1239–54
23. Lange Y, Ye J, Chin J. The fate of cholesterol exiting lysomes. J
Biol Chem 1997;272:17018–22
24. Lange Y, Ye J, Steck TL. Circulation of cholesterol between lysosomes and the plasma membrane. J Biol Chem 1998;273:18915–22
25. Underwood KW, Jacobs NL, Howley A, Liscum L. Evidence for a
cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane. J Biol Chem
1998;273:4266–74
J Neuropathol Exp Neurol, Vol 59, December, 2000
1116
XIE ET AL
26. Neufeld EB. What the Niemann-Pick type C gene has taught us
about cholesterol transport. In: Chang T-Y, Freeman DA, eds. Intracellular cholesterol trafficking. Norwell, MA: Kluwer Academic
Publishers, 1998:93107
27. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res 1995;36:211–28
28. Osono Y, Woollett LA, Marotti KR, et al. Centripetal cholesterol
flux from extrahepatic organs to the liver is independent of the
concentration of high density lipoprotein-cholesterol in plasma.
Proc Natl Acad Sci USA 1996;93:4114–19
29. Jolley CD, Woollett LA, Turley SD, Dietschy JM. Centripetal cholesterol flux to the liver is dictated by events in the peripheral organs and not by the plasma high density lipoprotein or apolipoprotein A-I concentration. J Lipid Res 1998;39:2143–49
30. Acton S, Rigotti A, Landschulz KT, et al. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science
1996;271:518–20
31. Landschulz KT, Pathak RK, Rigotti A, et al. Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor,
in liver and steroidogenic tissues of the rat. J Clin Invest 1996;98:
984–95
32. Woollett LA, Spady DK. Kinetic parameters for high density lipoprotein apoprotein AI and cholesteryl ester transport in the hamster. J Clin Invest 1997;99:1704–13
33. Ueda Y, Royer L, Gong E, et al. Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL
cholesterol in scavenger receptor class B type I transgenic mice. J
Biol Chem 1999;274:7165–71
34. Xie C, Turley SD, Dietschy JM. Centripetal cholesterol flow from
the extrahepatic organs through the liver is normal in mice with
mutated Niemann-Pick Type C protein (NPC1). J Lipid Res 2000
(in press)
35. Belknap WM, Dietschy JM. Sterol synthesis and low density lipoprotein clearance in vivo in the pregnant rat, placenta, and fetus.
J Clin Invest 1988;82:2077–85
36. Cavender CP, Turley SD, Dietschy JM. Sterol metabolism in fetal,
newborn, and suckled lambs and their response to cholesterol after
weaning. Am J Physiol 1995;269:E331–E340
37. Jurevics H, Morell P. Cholesterol for synthesis of myelin is made
locally, not imported into brain. J Neurochem 1995;64:895–901
38. Turley SD, Dietschy JM. Regional variation in cholesterol synthesis
and low density lipoprotein transport in the brain of the fetus, newborn and adult animal. Nutrition, Metabolism and Cardiovascular
Diseases 1997;7:195–201
39. Jurevics HA, Kidwai FZ, Morell P. Sources of cholesterol during
development of the rat fetus and fetal organs. J Lipid Res 1997;38:
723–33
40. Turley SD, Burns DK, Dietschy JM. Preferential utilization of newly synthesized cholesterol for brain growth in neonatal lambs. Am
J Physiol 1998;274:E1099–E1105
41. Turley SD, Burns DK, Rosenfeld CR, Dietschy JM. Brain does not
utilize low density lipoprotein-cholesterol during fetal and neonatal
development in the sheep. J Lipid Res 1996;37:1953–61
42. Posse de Chaves EI, Rusiñol AE, Vance DE, et al. Role of lipoproteins in the delivery of lipids to axons during axonal regeneration. J Biol Chem 1997;272:30766–73
43. Snipes GJ, McGuire CB, Norden JJ, Freeman JA. Nerve injury
stimulates the secretion of apolipoprotein E by nonneuronal cells.
Proc Natl Acad Sci USA 1986;83:1130–34
44. Weisgraber KH, Roses AD, Strittmatter WJ. The role of apolipoprotein E in the nervous system. Curr Opin Lipidology 1994;5:
110–16
45. Trommsdorff M, Gotthardt M, Hiesberger T, et al. Reeler/disabledlike disruption of neuronal migration in knockout mice lacking the
VLDL receptor and apoE receptor 2. Cell 1999;97:689–701
J Neuropathol Exp Neurol, Vol 59, December, 2000
46. Osono Y, Woollett LA, Herz J, Dietschy JM. Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse. J Clin Invest 1995;95:
1124–32
47. Ishibashi S, Brown MS, Goldstein JL, et al. Hypercholesterolemia
in low density lipoprotein receptor knockout mice and its reversal
by adenovirus-mediated gene delivery. J Clin Invest 1993;92:
883–93
48. Turley SD, Herndon MW, Dietschy JM. Reevaluation and application of the dual-isotope plasma ratio method for the measurement
of intestinal cholesterol absorption in the hamster. J Lipid Res 1994;
35:328–39
49. Turley SD, Andersen JM, Dietschy JM. Rates of sterol synthesis
and uptake in the major organs of the rat in vivo. J Lipid Res 1981;
22:551–69
50. Spady DK, Dietschy JM. Sterol synthesis in vivo in 18 tissues of
the squirrel monkey, guinea pig, rabbit, hamster and rat. J Lipid
Res 1983;24:303–15
51. Dietschy JM, Spady DK. Regulation of low density lipoprotein uptake and degradation in different animals species. In: Parnham MJ,
Brune K, eds. Agents and actions supplements. Stuttgart: Birkhäuser Verlag, 1984:177–90
52. Jeske DJ, Dietschy JM. Regulation of rates of cholesterol synthesis
in vivo in the liver and carcass of the rat measured using [3H]water.
J Lipid Res 1980;21:364–76
53. Cheng CM, Joncas G, Reinhardt RR, et al. Biochemical and morphometric analyses show that myelination in the insulin-like growth
factor 1 null brain is proportionate to its neuronal composition. J
Neurosci 1998;18:5673–81
54. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:
265–75
55. Wu Y-P, Kubota A, Suzuki K. Neuronal death and reactive glial
changes in the brain of the Niemann-Pick disease type C mouse.
Society for Neuroscience Abstracts 1999;25:1118
56. Henderson LP, Lin L, Prasad A, et al. Embryonic striatal neurons
from Niemann-Pick type C mice exhibit defect in cholesterol metabolism and neurotrophin responsiveness. J Biol Chem 2000;275:
20179–87
57. Handelmann GE, Boyles JK, Weisgraber KH, et al. Effects of apolipoprotein E, very low density lipoproteins, and cholesterol on the
extension of neurites by rabbit dorsal root ganglion neurons in vitro.
J Lipid Res 1992;33:1677–88
58. Holtzman DM, Pitas RE, Kilbridge J, et al. Low density lipoprotein
receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell
line. Proc Natl Acad Sci USA 1995;92:9480–84
59. Fagan AM, Bu G, Sun Y, et al. Apolipoprotein E-containing high
density lipoprotein promotes neurite outgrowth and is a ligand for
the low density lipoprotein receptor-related protein. J. Biol. Chem.
1996;271:30121–25
60. Nathan BP, Bellosta S, Sanan DA, et al. Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro. Science 1994;
264:850–52
61. Lütjohann D, Bruer O, Ahlborg G, et al. Cholesterol homeostasis
in human brain: Evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc Natl Acad
Aci USA 1996;93:9799–804
62. Björkhem I, Lütjohann D, Breuer O, et al. Importance of a novel
oxidative mechanism for elimination of brain cholesterol. J Biol
Chem 1997;272:30178–84
63. Lund EG, Guileyardo JM, Russell DW. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the
brain. Proc Natl Acad Sci USA 1999;96:7238–43
NEURAL CHOLESTEROL ACCUMULATION IN NPC DISEASE
64. Björkhem I, Lütjohann D, Diczfalusy U, et al. Cholesterol homeostasis in human brain: Turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation. J Lipid Res 1998;39:1594–1600
65. Wilson JD. The measurement of the exchangeable pools of cholesterol in the baboon. J Clin Invest 1970;49:655–65
66. Lütjohann D, Papassotiropoulos A, Björkhem I, et al. Plasma 24Shydroxycholesterol (cerebrosterol) is increased in Alzheimer and
vascular demented patients. J Lipid Res 2000;41:195–98
1117
67. Beffert U, Danik M, Krzykowski P, et al. The neurobiology of
apolipoproteins and their receptors in the CNA and Alzheimer’s
disease. Brain Res Rev 1998;27:119–42
68. Popko B, Goodrum JF, Bouldin TW, et al. Nerve regeneration occurs in the absence of apolipoprotein E in mice. J Neurochem 1993;
60:1155–58
Received April 26, 2000
Revision received August 14, 2000
Accepted August 15, 2000
J Neuropathol Exp Neurol, Vol 59, December, 2000