1400Journal of Lipid Research Volume 52, 2011
Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Niemann-Pick type C (NPC) disease is a heritable lyso-
somal storage disorder, in which the intracellular trans-
port of lipids is perturbed ( 1 ). The cellular phenotype of
NPC disease is massive accumulation of cholesterol and
other lipids in membranous organelles derived from late
endosomes and lysosomes ( 2–5 ). Since the “trapped” cho-
lesterol is not metabolically available, various regulatory
pathways sense an apparent shortage, and paradoxically,
denovo synthesis is increased, further exacerbating the
situation ( 6, 7 ). Although dysregulated lipid processing
occurs in most organ systems of NPC patients, the primary
pathology they present is localized to the central nervous
system, in the form of progressive neurodegeneration.
Specifi cally, NPC patients suffer from motor and coordi-
nation dysfunctions, seizures, and cognitive impairments
that typically present during the fi rst decade of life. NPC
disease is fatal, and most patients succumb to it before
reaching teen age (e.g., Refs. 1, 8, 9 ). Intensive investiga-
tions in the recent two decades have led to the develop-
ment of diverse therapeutic intervention strategies, most
of which aim to repair the imbalance in specifi c lipids or
metabolites ( 10 ). To date, however, only limited clinical
benefi t has been achieved, at best leading to stabilization
of clinical symptoms (e.g., Ref. 11 ). The molecular culprits
underlying NPC disease have been shown to be loss-of-
function mutations in either NPC1 or NPC2 proteins,
which reside in the lysosomal limiting membrane or lu-
men, respectively ( 12–15 ). Although the precise mecha-
nisms of action of these proteins are not fully understood,
it is generally accepted that NPC1 and NPC2 function in
sequence in removing free cholesterol from the lysosomal
lumen to the cytosol ( 16–21 ). While NPC-affected lyso-
Abstract Vitamin E (alpha-tocopherol) is the major lipid-
soluble antioxidant in many species. Niemann-Pick type C
(NPC) disease is a lysosomal storage disorder caused by mu-
tations in the NPC1 or NPC2 gene, which regulates lipid
transport through the endocytic pathway. NPC disease is char-
acterized by massive intracellular accumulation of unesterifi ed
cholesterol and other lipids in lysosomal vesicles. We exam-
ined the roles that NPC1/2 proteins play in the intracellular
traffi cking of tocopherol. Reduction of NPC1 or NPC2 ex-
pression or function in cultured cells caused a marked lyso-
somal accumulation of vitamin E in cultured cells. In vivo,
tocopherol signifi cantly accumulated in murine Npc1 -null
and Npc2 -null livers, Npc2 -null cerebella, and Npc1 -null cere-
bral cortices. Plasma tocopherol levels were within the nor-
mal range in Npc1 -null and Npc2 -null mice, and in plasma
samples from human NPC patients. The binding affi nity of
tocopherol to the purifi ed sterol-binding domain of NPC1
and to purifi ed NPC2 was signifi cantly weaker than that of
cholesterol (measurements kindly performed by R. Infante,
University of Texas Southwestern Medical Center, Dallas,
TX). Taken together, our observations indicate that func-
tionality of NPC1/2 proteins is necessary for proper bio-
availability of vitamin E and that the NPC pathology might
involve tissue-specifi c perturbations of vitamin E status. —
Ulatowski, L., R. Parker, C. Davidson, N. Yanjanin, T. J. Kelley,
D. Corey, J. Atkinson, F. Porter, H. Arai, S. U. Walkley, and
D. Manor. Altered vitamin E status in Niemann-Pick type C
disease. J. Lipid Res. 2011. 52: 1400–1410.
Supplementary key words nutrition • oxidized lipids • Niemann-Pick
This work was supported by National Institutes of Health Grants DK-067494
(D.M.) and HD-045561 (S.U.W.); Bench-to-Bedside Award (F.P.) from the Na-
tional Institutes of Health, Offi ce of Rare Diseases; and the Intramural Research
Program (F.P.) of the National Institutes of Health, National Institute of Child
Health and Human Development. Its contents are solely the responsibility of the
authors and do not necessarily represent the offi cial views of the National Insti-
tutes of Health or other granting agencies. N.Y. was supported by the Ara
Parseghian Medical Research Foundation (Tucson, AZ) and Dana Angel’s Re-
search Trust (Greenwich, CT).
Manuscript received 18 March 2011 and in revised form 4 May 2011.
Published, JLR Papers in Press, May 5, 2011
Altered vitamin E status in Niemann-Pick type C disease
L. Ulatowski , * R. Parker , ** C. Davidson , †† N. Yanjanin , §§ T. J. Kelley , † D. Corey , † J. Atkinson , ***
F. Porter , §§ H. Arai , ††† S. U. Walkley , †† and D. Manor 1 , * , §
Departments of Nutrition,* Pediatrics, † and Pharmacology, § School of Medicine, Case Western Reserve
University , Cleveland, OH ; Division of Nutritional Sciences,** Cornell University , Ithaca, NY ; Department of
Neuroscience, †† Rose F. Kennedy Center for Research in Mental Retardation and Human Development,
Albert Einstein College of Medicine , Bronx, NY ; Program in Developmental Endocrinology and Genetics, §§
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of
Health, Bethesda , MD ; Department of Chemistry,*** Brock University , St. Catharines, Ontario, Canada ; and
Department of Health Chemistry, ††† University of Tokyo, Tokyo, Japan
Abbreviations: CNS, central nervous system; IHH, immortalized
human hepatocytes; NPC, Niemann-Pick type C; shRNA, short hairpin
RNA; ROS, reactive oxygen species; TMS, trimethylsilyl ether; TTP,
? -tocopherol transfer protein.
1 To whom correspondence should be addressed.
by Jeffrey Atkinson, on July 5, 2011
NPC1/2 and vitamin E status1401
The Npc1 ? / ? mice (BABLc/ NPC nih ) were originally described
in Ref. 12 , and the Npc2 ? / ? mice were described in Ref. 41 . Hu-
man serum samples were collected from NPC1 patients and
healthy age-appropriate unaffected subjects under a clinical pro-
tocol (06-CH-0186) approved by the NICHD Institutional Review
Board of the National Institute of Child Health and Human De-
velopment. Both consent and assent, if appropriate, were ob-
tained. Serum samples were de-identifi ed and maintained at ? 80
Cells were plated on poly-L-lysine coated glass coverslips in
24-well tissue culture plates. NBD-cholesterol (Invitrogen) and
NBD-tocopherol ( 42, 43 ) were complexed to serum lipoprotein
as described earlier ( 35, 44 ) and added to the culture media to a
fi nal concentration of 20 ? M and incubated for 17 h at 37°C. The
fl uorescent lipid was “chased” by incubation in normal media for
3 h more. Cells were fi xed for 20 min in 3.7% paraformaldehyde
and mounted in SlowFade Gold antifade reagent (Invitrogen)
prior to imaging on a confocal or inverted fl uorescence micro-
scope (Zeiss LSM 510 and Leica DMI 4000B, respectively). For
quantitation of accumulated fl uorophores, ten microscopic im-
ages were captured under identical conditions, each containing
30-60 cells. Fluorescence intensities were quantitated using Im-
age J software (http://rsbweb.nih.gov/ij/index.html). The RGB
images were converted to an 8-bit images; a common threshold
set for all images. For colocalization studies, LysoTracker Red
DND-99 (75 nM, Invitrogen) was added 30 min prior to fi xing.
For visualization of free cholesterol, fi xed and permeabilized
cells were incubated with 25 ? g/ml fi lipin (Streptomyces fi lipin-
ensis; Sigma Chemical) for 1 h at room temperature in the dark,
prior to washing in PBS and visualization.
Total cholesterol. Cells were harvested, resuspended in PBS,
and lysed by repeated passing through a 22-gauge needle. Total
cholesterol was measured using the Amplex Red Cholesterol As-
say kit (Invitrogen) according to manufacturer’s protocol. Fluo-
rescence was excited at 530 nm and emission was collected at 590
nm on a Tecan GENios Pro plate reader (Tecan, Durham, NC).
Total cholesterol was normalized to total protein, as determined
by the Bio-Rad protein assay kit.
Tocopherols and free cholesterol. Serum and appropriate tis-
sues from Npc1 ? / ? and Npc2 ? / ? mice and their wild-type litter-
mates were freshly excised and fl ash-frozen as described previously
( 45 ). Lipids were extracted, silylated, and analyzed by GC-MS on
a Hewlett-Packard 6890 gas chromatograph coupled to a Hewlett-
Packard 5872 mass selective detector operated in selected ion
mode as previously described ( 46 ). Deuterated ? -tocopherol added
prior to extraction served as an internal standard. Monitored masses
of trimethylsilyl ethers (TMS) were 511.6 (d 9 - ? -tocopherol-TMS),
502.6 (d 0 - ? -tocopherol-TMS), 488.6 (d 0 - ? -tocopherol-TMS), and
458.7 (cholesterol-TMS). A previously determined detector re-
sponse correction factor was applied in quantitation of choles-
terol. Tocopherol and unesterifi ed cholesterol concentrations
were normalized to tissue wet weight.
Binding of tocopherol to purifi ed NPC1 and NPC2
The affi nity of ? -tocopherol to the purifi ed NPC1/2 proteins
was measured by Rodney Infante and Joseph Goldstein at the
University of Texas Southwestern Medical Center (Dallas, TX)
using a published assay based on competition with radio-labeled
cholesterol ( 17 ). Briefl y, 4 pmol purifi ed sterol binding domain
somes accumulate large amounts of free cholesterol, intra-
cellular transport of glycosphingolipids, sphingomyelin, and
sphingosine is also severely perturbed ( 22 ). Which of these
trapped molecules (or their metabolites) is the metabolic
root for the NPC pathology is presently unknown ( 22 ).
It is interesting to note that common pathological and
biochemical hallmarks are shared by NPC disease and de-
fi ciency in the dietary antioxidant vitamin E. First, in both
cases, the major site of dysfunction is the central nervous
system (CNS), and the major clinical presentation is cere-
bellar ataxia ( 23–25 ), accompanied by specifi c injury to
cerebellar Purkinje neurons ( 26, 27 ). Second, axonal
spheroids (focal swellings) are frequently observed in both
NPC disease ( 28 ) and in vitamin E defi ciency ( 29–31 ).
Similarly, pronounced hypomyelination is characteristic
of advanced-stage disease in both cases ( 32, 33 ). Finally,
modest supplementation with vitamin E has been reported
to result in a mild improvement in motor performance in
a mouse model of NPC disease ( 34 ). On the cellular level,
it has been established that uptake of vitamin E occurs via
endocytosis ( 35, 36 ) and that a signifi cant portion of the
vitamin is found in lysosomes ( 37 ). In light of these obser-
vations, we hypothesized that proper intracellular traffi cking
of vitamin E (and in turn, adequate antioxidant protection)
depends on timely egress from the lysosome and, there-
fore, on the functionality of NPC1/2. We describe here
our fi ndings regarding ? -tocopherol status in cells that ex-
press defective alleles or reduced expression of NPC1/2,
in various tissues from mice in which expression of NPC1/2
is disrupted and in plasma from human NPC patients.
MATERIALS AND METHODS
Human fi broblasts harboring the p.P237S and p.I1061T mis-
sense mutations in the NPC1 gene were obtained from Coriell
Cell Repository (GM03123; Camden, NJ) and grown in Eagle’s
minimum essential medium with Earle’s salts, 2 mM L-glutamine
and 15% fetal bovine serum at 37°C and 5% CO 2 ( 38 ). Control
human fi broblasts (CRL-2076) were obtained from American
Type Culture Collection (Manassas, VA). Immortalized human
hepatocytes (IHH) ( 39, 40 ) were a generous gift from R. Ray
(Saint Louis University, St. Louis, MO) and were cultured in
Dulbecco’s modifi ed Eagle’s medium (DMEM) supplemented with
5% calf serum. Lentiviral short hairpin RNA (shRNA) constructs
targeted against human NPC1, human NPC2, and a control
shRNA in the pLKO vector (Open Biosystems, Huntsville, AL)
were transfected into HEK293T cells using Lipofectamine-Plus
(Invitrogen, Carlsbad, CA). Culture media were harvested 24 and
48 h posttransfection, pooled, and pelleted by centrifugation at
100,000 g for 1.5 h. The pellet was resuspended in PBS and used
for polybrene-mediated (4 ? g/ml) transduction of IHH cells us-
ing standard protocols. Stable knockdown clones were selected
in media supplemented with puromycin (10 ? g/ml; Sigma
Chemical Co., St. Louis, MO) 48 h after transduction. Knock-
down effi ciency was evaluated by immunoblotting using antibodies
raised against NPC1 (Abcam, Cambridge, MA) or NPC2 (gener-
ous gift of Peter Lobel, Rutgers University, Rutgers, NJ). For eval-
uating the endogenous expression levels of the ? -tocopherol
transfer protein (TTP), samples were immunoblotted using the
A8E5 anti-TTP antibody (H. Arai).
by Jeffrey Atkinson, on July 5, 2011
1402 Journal of Lipid Research Volume 52, 2011
Generation and characterization of NPC1 and NPC2
knockdown hepatocyte cell lines
Although genetic defects in NPC1 and NPC2 lead to se-
vere accumulation of cholesterol in the liver ( 55 ), no he-
patocyte cell culture model is presently available to study
the disease. We therefore generated lentiviruses that en-
code shRNAs against the human NPC1 and NPC2 tran-
scripts, and used these reagents to generate IHH ( 39 ) in
which the expression of NPC1 or NPC2 is stably disrupted.
As shown in Fig. 2A , expression of NPC1 and NPC2 in the
stable “knockdown” cell lines was reduced by ? 50% and
90%, respectively, compared with IHH cells which express a
control shRNA. Since egress of ? -tocopherol from the liver
depends on the hepatic TTP ( 35, 56 ), we examined
whether expression levels of TTP are altered in NPC1/2
knockdown cells. Immunoblotting with anti-TTP anti-
bodies revealed that expression levels of TTP in these cells
was comparable to the levels observed in control IHH cells
(data not shown). As altered intracellular distribution of
cholesterol is the cellular hallmark of NPC disease ( 55 ),
we examined the levels and intracellular distribution of
cholesterol in the NPC1/2 “knockdown” cells. Fig. 2B
shows the amount of total cholesterol retained in these
cells, as determined by the Amplex Red colorimetric assay
kit. In both shNPC1 and shNPC2 cells, total cellular cho-
lesterol was increased by approximately 2-fold compared
with control cells. To examine the effects of NPC1/2 on
the intracellular distribution pattern of cholesterol, we
employed the fl uorescent fungal macrolide fi lipin, which
selectively binds to free (unesterifi ed) cholesterol in mem-
branes ( 57 ), and is a primary tool for diagnosing NPC dis-
ease ( 2, 58 ). In control IHH cells, fi lipin fl uorescence
outlined free cholesterol exclusively in the cells’ plasma
membranes ( Fig. 2C , left panel). In shNPC1 and shNPC2
cells, however, the fi lipin-staining pattern was markedly
different: First, intensity of the fl uorescence signal was
much higher compared with control cells, indicating sig-
nifi cant accumulation of free cholesterol. Second, fi lipin
(NTD) of NPC1, or 8 pmol purifi ed full-length NPC2 were incu-
bated overnight with 130 nmol [ 3 H]cholesterol at 4°C. The proteins
were then incubated with 6 ? M unlabeled competitor (cholesterol,
epicholesterol, 25-hydroxycholesterol, or dl- ? tocopherol), and
protein-bound radioactivity was measured after affi nity chroma-
tography with nickel-agarose and scintillation counting.
Statistical signifi cance of data was determined using unpaired
Student’s t -test. P values < 0.05 were taken as the threshold of
signifi cance. Data were analyzed and graphed using the IgorPro
software package (Wavemetrics, Inc., Portland, OR).
? -tocopherol accumulates in NPC-affected fi broblasts
The NPC1 and NPC2 proteins are residents of the lyso-
some that are required for proper transit of cholesterol
through the endocytic pathway ( 15, 47 ). Given that
sphingomyelin, glycosphingolipids, and phospholipids
also accumulate in NPC-affected lysosomes ( 2, 48–51 ),
we hypothesized that NPC1/2 proteins participate in the
endocytic processing of the lipid-soluble antioxidant
? -tocopherol (vitamin E). To visualize the intracellular
traffi cking of ? -tocopherol, we utilized NBD-tocopherol,
a fl uorescent analog that we previously characterized in
vitro ( 42, 43, 52, 53 ) and in vivo ( 35, 54 ). Using fl uores-
cence microscopy, we visualized the accumulation of
NBD-tocopherol in cultured fi broblasts isolated from an
NPC-affected patient (harboring the c.709C>T and
c.3182T>C substitutions in the NPC1 gene) and control
fi broblasts. As shown in Fig. 1 , control fi broblasts re-
tained very little NBD-tocopherol. However, NPC-fi bro-
blasts accumulated much higher (ca. 3-fold) levels of the
fl uorescent vitamin, appearing in a punctate, perinuclear
distribution pattern. These observations indicate that
egress of ? -tocopherol from the endocytic compartment
requires a functional NPC1 protein.
Fig. 1. NBD-tocopherol accumulates in human
NPC1 fi broblasts. Indicated fi broblasts were incu-
bated with serum-complexed NBD-tocopherol over-
night and “chased” in normal growth media for 3 h.
Fixed cells were imaged by fl uorescence microscopy.
A: Representative fl uorescence micrographs. Magni-
fi cation: 60×. B: Quantitation of fl uorescence inten-
sity of 10 images, each including at least 30 cells.
Asterisks denote signifi cant difference ( P > 0.05)
from control shRNA cells, as determined by Stu-
dent’s t -test.
by Jeffrey Atkinson, on July 5, 2011
NPC1/2 and vitamin E status1403
precomplexed to serum lipoproteins. The shNPC1 and
shNPC2 cells accumulated signifi cantly higher levels ( ? 3-
fold) of NBD-cholesterol compared with control hepato-
cytes ( Fig. 2D ).Taken together, these results indicate that
hepatocytes with disrupted expression of NPC1 or NPC2
display the established lipid-traffi cking defects that charac-
terize NPC disease. Therefore, we conclude that the stable
shRNA IHH cell lines are an appropriate model system for
staining was seen primarily within the hepatocytes, in a
punctate, perinuclear pattern (arrows in center and right
panels of Fig. 2C ). This pattern is essentially identical to
the lysosomal accumulation of free cholesterol in other
NPC1/2 cell types ( 38, 41 ). Finally, we examined the intra-
cellular fate of cholesterol that was taken up through en-
docytosis. Toward this end, we monitored the uptake of
the fl uorescent analog NBD-cholesterol ( 59–62 ) that was
Fig. 2. Characterization of human hepatocytes stably expressing shRNAs to NPC1 or NPC2. IHH cells ex-
pressing the indicated shRNA were generated by lentiviral transduction and antibiotic selection as detailed
in Materials and Methods. A: Expression of NPC1 and NPC2 was examined by Western blotting in lysates
from the indicated sublines. B: Cellular content of total (esterifi ed plus free) cholesterol in the different
sublines was measured using the Amplex Red kit. Shown are averages and standard deviations of three inde-
pendent experiments. C: Content and distribution of unesterifi ed cholesterol were determined by fi lipin
staining. Note that in control cells, free cholesterol is localized exclusively to the plasma membrane, whereas
shNPC sublines exhibit pronounced intracellular accumulation, appearing as perinuclear vesicles (white
arrows). Scale bar = 10 ? m. D: Accumulation of NBD-cholesterol was examined after overnight loading with
serum-complexed NBD-cholesterol as described in Materials and Methods. Ten fl uorescent images, each
containing 40-60 cells, were digitized and fl uorescence intensity determined using Image J software. Asterisks in
B and D denote signifi cant difference ( P > 0.05) from control shRNA cells, as determined by Student’s t -test.
by Jeffrey Atkinson, on July 5, 2011
1404 Journal of Lipid Research Volume 52, 2011
shown in Fig. 3C , the intracellular distribution pattern of
NBD-tocopherol colocalized with that of LysoTracker, an
established marker of the late endocytic/lysosomal com-
partment ( 63, 64 ). We concluded that functionality of
NPC1 and NPC2 is required for the egress of endocytosed
vitamin E from the endocytic compartment. Furthermore,
under conditions of NPC1/2 impairment, the majority of
tocopherol accumulates in lysosomes in a pattern similar
to that of NBD-cholesterol.
Tocopherol is a poor ligand for NPC1 and NPC2
To gain insights into the molecular mechanisms by
which NPC1/2 affect tocopherol traffi cking, we directly
measured the binding affi nity of these proteins for
? -tocopherol. Toward this end, we examined the effi cacy
of vitamin E in competing with [ 3 H]cholesterol for bind-
ing to purifi ed NPC2 or to purifi ed recombinant sterol
binding domain of NPC1 (residues 1-240) ( 17, 19 ). Under
saturating conditions (tocopherol:binding site molar ratio
= 1000), ? -tocopherol was able to displace only 30% and
50% of the [ 3 H]cholesterol bound to NPC2 and NPC1,
respectively ( Fig. 4 ), whereas unlabeled cholesterol dis-
placed >85% of the bound ligand. These results indicate
that the affi nity of ? -tocopherol to NPC proteins is 2-3 or-
ders of magnitude weaker than that of cholesterol. These
in vitro fi ndings are put into physiological perspective when
appreciating that, in vivo, concentrations of cholesterol
are 100- to 1000-fold higher than those of ? -tocopherol
( 65 ), and this ratio is likely higher in lysosomes ( 66 ).These
considerations suggest that ? -tocopherol is not likely to oc-
cupy a signifi cant fraction of the NPC1/NPC2 binding
pockets in lysosomes of intact cells. Thus, we concluded
that the accumulation of ? -tocopherol observed in the
NPC-defective cells is likely an indirect effect, secondary to
the signifi cant buildup of lipids and the extensive struc-
tural reconfi guration of the late endocytic compartment.
This conclusion is consistent with reports regarding other
lipids that do not directly bind to NPC1 or NPC2, but that
investigating the roles of NPC proteins in the intrahepato-
cyte traffi cking of lipids, including vitamin E.
Disrupted expression of NPC1/2 causes lysosomal
accumulation of vitamin E in IHH cells
To examine the involvement of NPC proteins in traf-
fi cking of ? -tocopherol, we “loaded” the different IHH
cell lines with serum-complexed NBD-tocopherol and
examined accumulation of the vitamin using fl uorescence
microscopy. As shown in Fig. 3A , NBD-tocopherol was effi -
ciently taken up by the cells and concentrated in a vesicu-
lar, perinuclear compartment, reminiscent of our previous
observations in human HepG2 and rat McARH-7777 hepa-
tocytes ( 35, 54 ). We quantitated fl uorescence intensity
in images from three independent experiments, and we
found that cells with reduced expression of either NPC1
or NPC2 accumulated ? 2-fold more NBD-tocopherol
compared with control cells. Next, we utilized confocal
fl uorescence microscopy to determine the intracellular
compartment in which NBD-tocopherol accumulates. As
Fig. 3. Intracellular accumulation of NBD-tocopherol in shNPC1
and shNPC2 IHH cells. Cells were “loaded” with NBD-tocopherol
as described in Materials and Methods, and intracellular distribu-
tion of the vitamin was examined using fl uorescence microscopy.
A: Representative NBD-fl uorescence images. B: Accumulation of
NBD-tocopherol. Cells were loaded with NBD-tocopherol, and
fl uorescence intensity was measured as described in Materials and
Methods. Asterisks denote signifi cant difference ( P > 0.05)
from control shRNA cells, as determined by Student’s t -test. C:
Confocal fl uorescence micrographs showing colocalization of NBD-
tocopherol (green) with the lysosomal marker LysoTracker (red).
Fig. 4. Binding of vitamin E to NPC1/2 in vitro. Binding of radio-
labeled cholesterol to the purifi ed proteins was measured in the
presence of the indicated competitors as described in Materials
and Methods. CHOL, cholesterol; EPI, epicholesterol; 25HC,
25-hydroxycholesterol; TOH, dl- ? -tocopherol. Shown are averages
and standard deviations of three experiments. See Ref. 17 for
by Jeffrey Atkinson, on July 5, 2011
NPC1/2 and vitamin E status 1405
increased ( Fig. 5G ). This could be explained by the fact
that lipid accumulation is balanced by lipid loss that ac-
companies neurodegeneration in this tissue ( 6, 45, 73, 79 ).
Plasma tocopherol and cholesterol levels in NPC-affected
mice and humans
Figure 6 shows the concentrations of tocopherol and
cholesterol in plasma samples from Npc1 ? / ? , Npc2 ? / ? , and
wild-type mice. In agreement with published reports ( 6 ),
plasma cholesterol values of Npc1 ? / ? mice were not signifi -
cantly different from wild-type animals ( Fig. 6A ). Similarly,
plasma vitamin E levels were unchanged in Npc1 ? / ? mice
( Fig. 6B ). In 12-week-old Npc2 ? / ? mice, however, plasma
levels of both cholesterol and tocopherol were elevated by
approximately 30%. Unlike in other tissues, however, the
increase in the two lipids was essentially identical, such
that the tocopherol:cholesterol ratio did not differ among
the different mouse models ( Fig. 6C ).
Lastly, we analyzed the plasma levels of tocopherol in a
cohort of 45 NPC1 patients and 20 age-appropriate con-
trol subjects. Total cholesterol levels in plasma samples
from NPC patients were <200 mg/dl; i.e., within the nor-
mal range for adults as defi ned by the American Heart As-
sociation ( 80 ). These values are similar to those reported
previously for NPC1 patients ( 81 ). Importantly, plasma
levels of ? -tocopherol and the most prevalent vitamin E
form in the US diet, ? -tocopherol, were within the clini-
cally normal range (12-50 ? M) ( 82 ). Figure 7A and B show
the concentrations of ? - and ? -tocopherol, respectively,
after normalization to plasma cholesterol levels. Taken to-
gether, our data indicate that although NPC-affected cells
and tissues showed signifi cant alterations in the status of
vitamin E and cholesterol, plasma levels were not affected
in NPC-affected mice and humans.
Niemann-Pick type C disease is a debilitating, fatal disor-
der in which intracellular lipid transport is impaired due
to loss-of-function mutations in the NPC1 or NPC2 pro-
tein. The main biochemical phenotype associated with
NPC disease is accumulation of unesterifi ed cholesterol
and other lipids in a vesicular compartment of an endo-
somal/lysosomal origin. A number of metabolic scenarios
can be envisioned to be at the root of NPC pathology.
First, the extensive localized accumulation of lipids may be
toxic, thereby compromising cell function and viability.
Second, since the affected lipids are “sequestered” away
from their proper sites of action, the affected cell may ex-
perience a catastrophic defi ciency of these metabolites.
Lastly, physical disruption of the endocytic compartment
may deprive the cell of other molecules that rely on this
pathway for cellular transport. Despite intense research ef-
forts in the past 50 years, many questions regarding the
etiology of NPC disease remain unanswered. Thus, it is still
not known which of scenarios described above is of highest
signifi cance during disease progression. Similarly, it has
not been conclusively determined which of the lipids
sequestered in NPC lysosomes is the primary culprit re-
accumulate under NPC1 or NPC2 loss-of-function ( 21, 67,
Vitamin E status in NPC-affected mice
To examine the involvement of NPC proteins in the sta-
tus of vitamin E in vivo, we employed GC-MS to determine
the tocopherol content in extracts from plasma, livers, and
brains of Npc1 ? / ? and Npc2 ? / ? mice ( 41 ). To frame our
fi ndings in the context of overall lipid status, we also deter-
mined the free cholesterol content of these extracts. In
the liver, both cholesterol and tocopherol accumulated in
NPC-affected mice to higher levels than in wild-type ani-
mals. Specifi cally, hepatic concentrations of cholesterol
increased by 10- and 6-fold in the livers of 12-week old
Npc1 ? / ? and Npc2 ? / ? mice, respectively ( Fig. 5A ). These
values are similar to the hepatic values reported earlier for
these models ( 41, 55, 51, 68 ). Analyses of vitamin E content
revealed that hepatic levels of tocopherol also increased in
12-week-old NPC-affected mice, albeit to a lesser degree
( Fig. 5A ). Since vitamin E shares with cholesterol many
common uptake and transport steps, it is also instructive to
present the concentration values as tocopherol:cholesterol
mole ratios (see Refs. 69 and 70 for detailed discussion).
As seen in Fig. 5E , the tocopherol:cholesterol ratio was sig-
nifi cantly decreased in NPC-affected livers. Thus, while
NPC-affected livers accumulated both lipids, hepatic accu-
mulation of cholesterol exceeded that of tocopherol by >5-
fold. As a result, the effective disruption in hepatic vitamin E
status caused by NPC is actually more severe than appears
at fi rst sight. Mechanistically, such disproportionate accu-
mulation of the two lipids is likely to refl ect additional,
vitamin E-specifi c routes of egress from the endocytic
pathway that are not shared by cholesterol. The existence
of such secretion pathways is supported by the rapid turn-
over of hepatic tocopherol in plasma (approximately 1 he-
patic pool per day) ( 71 ) and by our observations that in
cultured hepatocytes, some NBD-tocopherol colocalizes
with the rapidly recycling, transferrin-positive compart-
ment (J. Qian and D. Manor, unpublished observations).
We also found that expression levels of TTP did not dif-
fer among the wild-type, Npc1 ? / ? , and Npc2 ? / ? mice ( Fig.
5D ). We concluded that accumulation of tocopherol in
NPC-affected mice does not stem from altered TTP ex-
pression but, rather, is a consequence of impairment in
the function of NPC1/2 proteins. In the cortex, we ob-
served a signifi cant (40-50%) decrease in the content of
tocopherol as well as cholesterol in 12-week-old Npc1 ? / ?
mice ( Fig. 5B ). We attributed this decrease to the severe
hypomyelination of the cortex that accompanies NPC dis-
ease ( 28, 41, 72 ). Both cholesterol and tocopherol are im-
portant constituents of myelin ( 73–75 ), and vitamin E
defi ciency causes hypomyelination ( 76–78 ). In the cere-
bellum, the only statistically signifi cant difference was ob-
served in 12-week-old Npc2 ? / ? mice, which exhibited a
? 30% increase in the content of tocopherol as well as cho-
lesterol, compared with wild-type animals ( Fig. 5C ). No
signifi cant differences in tocopherol or cholesterol con-
tent were observed in Npc1 ? / ? mice, although the frac-
tional lipid content (mole ratio) of tocopherol was slightly
by Jeffrey Atkinson, on July 5, 2011
1406 Journal of Lipid Research Volume 52, 2011
Fig. 5. Tocopherol and unesterifi ed cholesterol content in tissue extracts from Npc1 ? / ? , Npc ? / ? , and wild-type mice. Analytes were mea-
sured using GC-MS as described in Materials and Methods. A, E: Liver. B, F: Cerebral cortex. C, G: Cerebellum. Shown are averages and
standard deviations (n = 3). Asterisks denote signifi cant difference ( P < 0.05) compared with age-matched controls, as determined by Stu-
dent’s t -test. D: Expression levels of the ? tocopherol transfer protein in livers of the different mouse models. Expression levels were evalu-
ated by anti-TTP Western blotting of soluble extracts prepared from three animals of the indicated genotypes. WT, wild-type.
by Jeffrey Atkinson, on July 5, 2011
NPC1/2 and vitamin E status1407
We showed here that tocopherol is sequestered in vesi-
cles of lysosomal origin in NPC-affected fi broblasts and
hepatocytes. Furthermore, we showed that vitamin E status
is perturbed in brains and livers of Npc1 ? / ? and Npc2 ? / ?
mice. Thus, it is possible that imbalance in vitamin E status
contributes to the progression of NPC disease, and con-
versely, that supplementation with ? -tocopherol may ben-
efi t those affl icted with this disorder. Although vitamin E
supplementation was reported in Npc1 ? / ? mice ( 34 ), the
measured endpoints were limited, and no study of such
supplementation has been reported in human patients.
It is important to note that concentrations of vitamin E
in plasma samples from NPC-affected mice and humans
were not signifi cantly different from those of healthy con-
trols. The immediate implication of these fi ndings is that
plasma tocopherol concentrations do not refl ect vitamin E
status in tissues and cells and, thus, are of limited clinical
use. This is not the fi rst time such a concern has been
raised. Sokol et al. studied a small pediatric cholestasis co-
hort and found that in some cases vitamin E defi ciency
occurs in the presence of “normal” plasma tocopherol lev-
els ( 87 ). On the mechanistic level, these fi ndings may be
explained by the presence of homeostatic mechanisms
that maintain constant circulating levels of tocopherol, de-
spite severe localized perturbations in specifi c tissues and
cells, similar to the regulation of plasma cholesterol. On
the practical level, these observations raise the urgent
need for an adequate biomarker that refl ects tissue vita-
sponsible for NPC pathology (see Ref. 22 for discussion).
Moreover, the detailed biochemical mechanisms of action
of the NPC1 protein are still enigmatic.
The neurological hallmarks of NPC disease share strik-
ing similarity to those presented during vitamin E defi -
ciency. On the clinical level, the primary presentation of
both diseases is ataxia, refl ecting selective injury of cere-
bellar Purkinje neurons. On the microscopic level, the two
pathologies share the presence of axonal swellings (spher-
oids) and hypomyelination. These associations raise the
possibility that oxidative stress is a signifi cant factor con-
tributing to the etiology of NPC disease. Indeed, NPC-
affected cells exhibit mitochondrial dysfunctions ( 83 ),
increased expression of reactive oxygen species (ROS)-
producing and oxidative stress-responsive genes ( 84 ), and
elevated plasma levels of oxidized cholesterol ( 85 ). Recent
studies demonstrated that plasma samples from human
NPC patients exhibit compromised ex-vivo antioxidant ca-
pacity ( 86 ). Our fi ndings confi rm and extend these obser-
vations with regards to the lipophilic antioxidant vitamin E.
Fig. 6. Plasma tocopherol levels are normal in Npc1 ? / ? and
Npc2 ? / ? mice and NPC-affected humans. Tocopherol and cholesterol
levels were measured in plasma samples of the indicated mouse
models using GC-MS as described in Materials and Methods. Shown
are averages and standard deviations (n = 3). Asterisks denote sig-
nifi cant difference ( P < 0.05) compared with age-matched controls,
as determined by Student’s t -test.
Fig. 7. Plasma vitamin E:cholesterol ratios are normal in human
NPC1 patients. Plasma was collected from 45 NPC1 patients
and 20 healthy age-appropriate controls, and concentrations of
? -tocopherol (A) and ? -tocopherol (B) as well as unesterifi ed choles-
terol were determined using GC-MS as described in Materials and
Methods. Data are represented in a box plot, in which the horizon-
tal line designates the median value, separating the upper and
lower quartiles. The “whiskers” show the maximum and minimum
spread of the data.
by Jeffrey Atkinson, on July 5, 2011
1408 Journal of Lipid Research Volume 52, 2011
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min E status. Since analysis of the NPC-affected tissue is
impractical, a sensitive circulating indicator of oxidative
stress may be the appropriate biomarker in this case. Cir-
culating unsaturated lipid peroxidation products, such as
HETEs and isoprostanes ( 88, 89 ), and hydroxylated cho-
lesterol metabolites ( 85 ) may serve as the proper bio-
marker for these purposes. Clearly, there is a dire need to
better defi ne and optimize the most suitable plasma marker
for oxidation status and to streamline its applicability for
routine clinical use.
Our data indicate that CNS tocopherol status is adversely
affected in NPC disease. In light of the relative ease, low
cost, and lack of ill effects associated with moderate vitamin
E supplementation, our observations support the design
of a clinical trial in which the clinical benefi t of vitamin E
supplementation will be assessed in NPC patients.
The authors thank T. Y. Chang, Laura Liscum, Peter Lobel, and
members of their labs for invaluable advice and reagents. The
authors thank the Hadley Hope Fund and Ed Cutler
(Phlebotomy Services International) for their assistance in
obtaining samples from control subjects. The authors would
also like to acknowledge the contribution of the caretakers and
patients who participated in this study.
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by Jeffrey Atkinson, on July 5, 2011