Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts.

Abstract

Niemann-Pick type C (NPC) disease is predominantly caused by mutations in the NPC1 protein that affect intracellular cholesterol trafficking and cause accumulation of unesterified cholesterol and other lipids in lysosomal storage organelles. We report the use of a series of small molecule histone deacetylase (HDAC) inhibitors in tissue culture models of NPC human fibroblasts. Some HDAC inhibitors lead to a dramatic correction in the NPC phenotype in cells with either one or two copies of the NPC1(I1061T) mutation, and for several of the inhibitors, correction is associated with increased expression of NPC1 protein. Increased NPC1(I1061T) protein levels may partially account for the correction of the phenotype, because this mutant can promote cholesterol efflux if it is delivered to late endosomes and lysosomes. The HDAC inhibitor treatment is ineffective in an NPC2 mutant human fibroblast line. Analysis of the isoform selectivity of the compounds used implicates HDAC1 and/or HDAC2 as likely targets for the observed correction, although other HDACs may also play a role. LBH589 (panobinostat) is an orally available HDAC inhibitor that crosses the blood-brain barrier and is currently in phase III clinical trials for several types of cancer. It restores cholesterol homeostasis in cultured NPC1 mutant fibroblasts to almost normal levels within 72 h when used at 40 nM. The findings that HDAC inhibitors can correct cholesterol storage defects in human NPC1 mutant cells provide the potential basis for treatment options for NPC disease.

Full text

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Histone deacetylase inhibitor treatment dramatically
reduces cholesterol accumulation in Niemann-Pick
type C1 mutant human fibroblasts
Nina H. Pipaliaa, Casey C. Cosnerb, Amy Huanga, Anamitra Chatterjeeb, Pauline Bourbonb, Nathan Farleyb,
Paul Helquistb,1, Olaf Wiestb,1, and Frederick R. Maxfielda,1,2
aDepartment of Biochemistry, Weill Cornell Medical College, New York, NY 10065; andbDepartment of Chemistry and Biochemistry, University of Notre
Dame, Notre Dame, IN 46556
Edited* by Matthew P. Scott, Stanford University/Howard Hughes Medical Institute, Stanford, CA, and approved February 24, 2011 (received for review
October 4, 2010)
Niemann-Pick type C (NPC) disease is predominantly caused by
mutations in the NPC1 protein that affect intracellular cholesterol
trafficking and cause accumulation of unesterified cholesterol and
other lipids in lysosomal storage organelles. We report the use of
a series of small molecule histone deacetylase (HDAC) inhibitors in
tissue culture models of NPC human fibroblasts. Some HDAC
inhibitors lead to a dramatic correction in the NPC phenotype in
cells with either one or two copies of the NPC1I1061Tmutation, and
for several of the inhibitors, correction is associated with increased
expression of NPC1 protein. Increased NPC1I1061Tprotein levels
may partially account for the correction of the phenotype, because
this mutant can promote cholesterol efflux if it is delivered to late
endosomes and lysosomes. The HDAC inhibitor treatment is inef-
fective in an NPC2 mutant human fibroblast line. Analysis of the
isoform selectivity of the compounds used implicates HDAC1 and/
or HDAC2 as likely targets for the observed correction, although
other HDACs may also play a role. LBH589 (panobinostat) is an
orally available HDAC inhibitor that crosses the blood–brain bar-
rier and is currently in phase III clinical trials for several types of
cancer. It restores cholesterol homeostasis in cultured NPC1 mu-
tant fibroblasts to almost normal levels within 72 h when used at
40 nM. The findings that HDAC inhibitors can correct cholesterol
storage defects in human NPC1 mutant cells provide the potential
basis for treatment options for NPC disease.
H
their regulatory role in chromatin remodeling and gene tran-
scription (1). These enzymes function in a synchronized feedback
loop mechanism to mediate posttranslational acetylation and
deacetylation of many types of proteins including histones,
transcription factors, and chaperones, such as Hsp90 (2). The 18
HDACs that have been identified in mammals can be divided
into four distinct classes based on their homology to yeast pro-
teins (3, 4). Class I HDACs include HDAC1, -2, -3, and -8. Class
II is divided in two subclasses, with IIa (containing one HDAC
domain) including HDAC4, -5, -7, and -9 and class IIb (with two
HDAC domains) including HDAC6 and -10. The only known
representative of class IV in humans is HDAC11. Class III
HDACs are structurally distinct in that they are homologous to
yeast Sir2 and have NAD-dependent deacetylase activity. Al-
though most HDACs reside in the nucleus, some are also found
to shuttle between the nucleus and the cytoplasm (5).
Although the specific function of individual isoforms of HDAC
or the mechanisms leading to the observed effects of inhibition
are not clearly understood (6), studies have shown their diverse
roles in cell proliferation, cell death (7), and tissue-specific de-
velopmental activity (8). This wide range of activities, together
withthefactthatHDACshavebeenfoundtobedruggabletargets
for cancer and many other disorders (7, 9), has led to an intense
research effort, including the development of inhibitors to regu-
late their activity. Current HDAC inhibitors (HDACi) belong
istone acetyl transferases (HAT) and histone deacetylases
(HDAC) have gained considerable recognition because of
to one of four structural classes: hydroxamates, cyclic peptides,
aliphatic acids, or benzamides. Two HDACi [suberoylanilide
hydroxamic acid (SAHA) and FK-228] have been approved for
pharmaceutical use in the United States, and more than 10 are in
clinical trials (10).
Niemann-Pick disease type C (NPC) is a fatal neurodegener-
ative lysosomal storage disorder resulting in abnormal accumu-
lation of unesterified cholesterol, glycosphingolipids, and other
lipidsinlateendosome/lysosomes(LE/Ly)ofmanycelltypes.The
incidence is estimated between 1:120,000 and 1:50,000 live births
(11). In addition to the CNS, abnormal lipid accumulation occurs
in peripheral organs, leading to pathology in these tissues. Two
genes,npc1andnpc2,havebeenlinkedtotheNPCdefect,andthe
precise mechanisms of action of these proteins are under in-
vestigation. NPC1 is a multispanning transmembrane protein that
islocalizedinthelimitingmembraneoftheLE/Ly(12),andNPC2
is a soluble protein that is found in LE/Ly and is able to bind
cholesterol (13). NPC2 has been shown to shuttle free cholesterol
to and from membranes in vitro and to the N-terminal choles-
terol-binding domain of NPC1 (14, 15).
The NPC1I1061Tmutation, which is expressed in the NPC1
mutant fibroblasts used in this study, is the most common muta-
tion observed in NPC1 patients and represents 15–20% of all
disease alleles (16, 17). NPC1 expression is subject to post-
transcriptional regulation, and it was observed that NPC1I1061T
protein is expressed at much lower levels in NPC1 fibroblasts
compared with NPC1 in WT cells (18). Studies on the processing
and stability of the NPC1I1061Tmutant protein in human fibro-
blastsshowedthat,althoughtheNPC1I1061Tproteinissynthesized
normally, it fails to undergo normal posttranslational glycosyla-
tion (19). Much of the NPC1I1061Tprotein is a misfolded protein
in the endoplasmic reticulum (ER), and it is subjected to pro-
teasomal degradation. The overexpression of NPC1I1061Tor use
of chemical chaperones rescues the NPC1 phenotype, indicating
that the mutant is functional if delivered to LE/Ly (19).
Treatment options for NPC disease are limited. The only drug
approved for treatment of NPC disease is Zavesca (Miglustat),
which inhibits glycosphingolipid synthesis (20). This treatment
slows the disease progression, but it does not reverse the dam-
aged neurons or promote recovery of lost neurons. Therapy us-
Author contributions: N.H.P., P.H., O.W., and F.R.M. designed research; N.H.P., C.C.C.,
A.H., A.C., P.B., N.F., and O.W. performed research; N.H.P., C.C.C., A.C., P.B., N.F., and
P.H. contributed new reagents/analytic tools; N.H.P., A.H., P.H., O.W., and F.R.M. analyzed
data; and N.H.P., P.H., O.W., and F.R.M. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1P.H., O.W., and F.R.M. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: frmaxfie@med.cornell.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1014890108/-/DCSupplemental.
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ing 2-hydroxyl propyl cyclodextrin as a cholesterol transporter,
which is delivered to LE/Ly and bypasses the need for NPC1 or
NPC2 (21, 22), is another option that is effective in reversing the
defect in cell culture and mouse and cat models (23). A limited
initial human trial of cyclodextrin is currently in progress.
There has been a suggestion that there may be a connection
between histone hyperacetylation and the level of NPC1 mRNA
in response to cAMP (24). More recently, it was shown that
cholesterol homeostasis in NPC1−/−mice was improved by
treatment with valproic acid, a very weak HDACi (25). However,
at the millimolar concentrations used, valproic acid is known to
have a large number of effects and targets (26). HDACi increase
the acetylation level of several nonhistone proteins, such as
transcription factors, cytoskeletal proteins, and molecular chap-
erones (27). Therefore, the potential mechanism of HDAC in-
hibition on the NPC phenotype is not well-understood. Starting
from our previous interest in HDACi (7, 28) and NPC disease
(21, 29, 30), we investigated the effect of HDACi on cholesterol
homeostasis in human NPC mutant fibroblasts.
Results
From the wide range of available HDACi, we synthesized or
purchased a collection of six HDACi (Table 1 and Fig. S1) se-
lected to provide a range of chemotypes, potencies, and selectiv-
ities: a moderately potent benzamide, CI-994; the United States
Food and Drug Administration (FDA)-approved hydroxamic
acid, SAHA; the potent hydroxamic acids, LBH589 and trichos-
tatin A (TSA); and two isoform-selective HDACi, thiophene
benzamide (31) and PCI-34051 (32). Human NPC1 mutant fi-
broblast cells GM03123 (a compound heterozygote with one al-
lele with a P237S mutation and a second allele with an I1061T
mutation) were initially treated with HDACi for 48 h at various
concentrations. Free (i.e., unesterified) cholesterol levels in ly-
sosomal storage organelles (LSOs) were determined quantita-
tively using an automated microscopy analysis based on the
binding of the fluorescent dye, filipin, to cholesterol as described
previously (30). Representative images of untreated filipin-
stained WT cells (GM05659) (Fig. 1A) indicate no significant
accumulation of free cholesterol in the LE/Ly. Filipin staining of
GM03123 NPC1 mutant human fibroblast cells, treated with the
vehiclecontrol DMSO(Fig.1B),shows extensive accumulation of
free cholesterol in the LSOs. Most interestingly, the treatment of
the GM03123 cells with either LBH589 (40 nM) (Fig. 1C) or TSA
(120 nM) (Fig. 1D) resulted in dramatic correction of the NPC1
phenotype as observed by reduced filipin staining in the LSOs.
The filipin labeling of unesterified cholesterol in HDACi-treated
NPC1 mutant cells was comparable with filipin labeling of WT
human fibroblasts.
The effect of each HDACi on GM03123 NPC1 mutant human
fibroblasts was determined at varying concentrations and times.
Fig. 2 shows the effects of four HDACi after 4, 24, 48, and 72 h.
All data are normalized to their matched DMSO controls. None
of the HDACi had a large effect within 4 h, but they all showed
dose-dependent effects after 1 d and greater effects after 2–3 d.
For CI-994 and SAHA, the EC50for treatment of GM03123 cells
was about 0.5 μM. For TSA, the EC50was about 50 nM. LBH589
was the most potent compound, and it corrects the NPC1 phe-
notype with an EC50below 5 nM. For both TSA and LBH589, the
filipin labeling of the LSOs was indistinguishable from the WT
cells at the optimal concentrations and 72 h of treatment. The
effectiveness of TSA and LBH589 was reduced at higher con-
centrations, probably reflecting interaction with multiple targets
at high doses. In summary, several HDACi are effective in cor-
recting the NPC1 mutant phenotype, and LBH589 was the most
potent inhibitor.
Encouraged by the effect of HDACi on NPC1 human fibro-
blasts from a donor carrying a heterozygous mutation, we in-
vestigated their efficacy on a different human NPC1 mutant
fibroblast line, GM18453, in which both alleles carry the I1061T
mutation. We also examined the HDACi effects on an NPC2
mutant human fibroblast line, GM18445, in which the donor was
homozygous, with both NPC2 alleles carrying a V39M mutation
as well as H215R and I858V homozygous polymorphisms in
Table 1.
mutant cells
Inhibition constants Ki(μM) for the HDACi against HDAC 1–9 compared with EC50(μM) in GM03123 NPC1
Compound\HDAC123456789EC50LSO*
CI-994†
SAHA†
TSA†
LBH-589‡
Thiophene benzam‡
PCI-34051§
0.05
0.0013
0.0002
0.001
0.007
4.0
0.19
0.0016
0.00065
0.00065
0.049
>50
0.55
0.005
0.005
0.0011
10
>50
>20
>20
1.4
0.55
>10
—
>20
3.6
0.26
0.08
>10
—
>20
0.0016
0.001
0.0015
>10
2.9
>20
>20
0.195
4.55
>10
—
>20
0.48
0.045
0.105
>10
0.01
>20
>20
0.8
3.2
—
—
0.5
0.5
0.05
<0.005
0.4
>10
*Approximate concentration required for a 50% reduction in LSO filipin labeling after 48–72 h (Fig. 2 and Fig. S5).
†Ref. 35.
‡Ref. 31.
§Ref. 32.
Fig. 1.
fibroblasts. NPC1 mutant human fibroblasts GM03123 were treated with
HDACi LBH589 or 120 TSA and incubated for 48 h. Cells were subsequently
fixed, stained with filipin, and imaged by epifluorescence microscopy at 10×
magnification. (A) WT human fibroblasts GM05659. (B) NPC1 human fibro-
blasts GM03123 treated with DMSO. (C) GM03123 cells after 48-h treatment
with 40 nM LBH589. (D) GM03123 cells after 48-h treatment with 120 nM
TSA. (Scale bar, 25 μM.)
Representative filipin images of HDACi-treated NPC1 mutant
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NPC1. The dose dependence after 48-h treatment with four
HDACi is shown in Fig. 3. Similar to the effect of HDACi on
GM03123, significant correction of the NPC1 phenotype was
observed on GM18453 NPC1 mutant cells. LBH589 was re-
markably effective in correcting the NPC1 phenotype at 5 nM,
with the highest efficacy at 40 nM (Fig. 3A). TSA was maximally
effective at 120 nM, and both CI-994 and SAHA caused im-
provement in the phenotype at concentrations above 370 nM.
Interestingly, none of the four HDACi tested were effective in
correcting the NPC phenotype in GM18445 NPC2 mutant cells
(Fig. 3B). Cell proliferation and cytotoxicity studies (Figs. S2 and
S3) indicate that the compounds are cytostatic but not cytotoxic
at the concentrations used.
We hypothesized that the mechanism of restoration of choles-
terol homeostasis by HDACi might be due to the increase NPC1
expression. Protein expression levels were measured after treat-
ment with three HDACi (SAHA, LBH589, and TSA) at their re-
spective optimum concentrations. Fig. 4 shows analysis of NPC1
proteinlevelsinWTcells(GM05659)andGM03123andGM18453
NPC1 mutantcells either untreated ortreated withDMSO,SAHA
(10 μM), LBH589 (40 nM), or TSA (370 nM) for 48 h. NPC1
protein expression is increased after HDACi treatment in both the
GM03123 and GM18453 cells. Thus, the correction of the NPC1
phenotype after HDACi treatment may be attributed, at least in
part, to increased expression of NPC1 protein.
We examined the cellular mechanisms for the reduction of
cholesterol in the LSOs of HDACi-treated NPC1 mutant cells.
The uptake of LDL was reduced in GM03123 NPC1 mutant
fibroblasts when they were treated with each of the HDACi at
their optimal concentration for 48 h (Fig. 5A and Fig. S4). The
untreated NPC1 mutant cells took up more LDL than WT
GM05659human fibroblasts, but the uptake approached the level
in WT cells after treatment. Expression of the LDL receptor is
regulated by sensing of cholesterol levels in the ER through the
sterol-responsive element binding protein-2 (SREBP2) tran-
scription factor, which is retained in the ER at high cholesterol
levels but delivered to the Golgi and proteolyzed to release the
active transcription factor at low cholesterol (33). Release of
cholesterol from LSOs should restore normal delivery of choles-
terol to the ERand reduce the proteolytic processing of SREBP2.
As shown in Fig. 5 B and C, treatment of NPC1 mutant cells with
HDACi for 18 h reduces the proteolytic processing of SREBP2,
consistent with improved delivery of cholesterol to the ER.
Release of cholesterol from LE/Ly and delivery to the ER
should also lead to increased cholesterol esterification by acyl
Co-A:cholesterol acyl transferase (ACAT), an ER enzyme. We
measured [14C]-oleic acid incorporation into cholesteryl-[14C]-
oleate as described (34). NPC1 mutant fibroblasts GM03123
were treated with HDACi at their most effective concentration
for 48 h. LBH589 treatment (40 nM) resulted in a 2.5-fold in-
crease in ACAT-mediated esterification compared with DMSO-
treated control cells (Fig. 5D). The induced esterification was
blocked by ACAT inhibitor 58–035. SAHA, CI-994, and TSA
treatment did not show a statistically significant effect. The
variability in the ACAT effects may be caused by the timing of
the measurement. After the HDACi treatment clears the cho-
lesterol in the LSOs and reduces LDL uptake, it would reduce
the amount of cholesterol available for esterification.
TofurtherprobethemechanismofHDACiaction,weexamined
which HDAC classes or isoforms might be responsible for the
observed effect. Hydroxamic acids and benzamides are relatively
weakinhibitorsofclassIIaHDACs.AsshowninTable1,LBH589,
Fig. 2.
(D) TSA at various concentrations and for varying time followed by fixing, staining with filipin, and imaging. DMSO was used as a solvent control. Images were
analyzed to obtain filipin fluorescence power in bright spots (filipin LSO ratio), and data were normalized to corresponding DMSO-treated cells. Data shown
are averages of three independent experiments totaling 60 images (five wells × four sites × three experiments). The dotted horizontal lines indicate mean
values for the solvent control; error bars represent SE.
Dose- and time-dependence plots for HDACi (set 1). NPC1 mutant human fibroblasts GM03123 were treated with (A) CI-994, (B) SAHA, (C) LBH589, or
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the most potent compound in our assay, has a Kiof 1 nM to
HDAC1but4.5μMtoHDAC7(35).Similarly,benzamidessuchas
CI-994 are very weak inhibitors of both class IIa and class IIb
HDACs (Kivalues > 20 μM). The function of class IV HDACs is
presentlyunknown,andtherearenoknowninhibitorsavailablefor
them. Therefore, class I HDACs are left as putative targets to be
studied. We tested two known isoform-selective inhibitors, PCI-
34051 (an HDAC8-selective inhibitor) (32) and thiophene benza-
mide (anHDAC1/2-selectiveinhibitor) (31), on two NPC1 mutant
human fibroblasts, GM03123 and GM18453. As shown in Fig. S5,
there is no correction of the NPC1 phenotype by the HDAC8-
selectiveinhibitor PCI-34051 in the 40 nM to 10 μM concentration
range. In contrast, treatment with the HDAC1/2-selective inhi-
bitor thiophene benzamide resulted in significant reduction of
cholesterol accumulation in LSOs of both NPC1 mutant human
fibroblast lines. These results suggest that HDAC8 is not the rel-
evanttargetofless-selectiveHDACi,whereasHDAC1-3inhibition
may play a role in reducing the NPC phenotype.
Discussion
We report here that treatment of NPC1 mutant human fibro-
blasts with some HDACi at nanomolar to micromolar concen-
trations leads to a nearly complete clearance of the excess
accumulation of cholesterol (Figs. 1–3). The lack of an effect
on NPC2 mutant cells (Fig. 3B) indicates that the mechanism
of action is not a general bypass of the NPC1- and NPC2-
dependent pathways as is seen in cells treated with cholesterol-
chelating cyclodextrins (21, 22). One component of the correc-
tion of the NPC phenotype is increased expression of the NPC1
protein (Fig. 4). There are several different mutations in the
human NPC1 protein that are associated with NPC disease (11).
The most common mutation, NPC1I1061T, exits the ER in-
efficiently and is mostly degraded (19). When chaperone activity
is enhanced, the NPC1I1061Tprotein can be delivered to LE/Ly,
where it has sufficient activity to reverse the NPC phenotype in
cultured cells (19). It is likely that several other NPC1 missense
mutations would also function if sufficient amounts of the pro-
tein could pass through the ER quality control processing. We
have shown that there is decreased cleavage of SREBP2 in the
HDACi-treated NPC1 mutant cells. This would be expected as
a consequence of release of the excess cholesterol from storage
organelles as NPC1 activity is increased. The reduced SREBP2
cleavage should reduce synthesis of LDL receptors, decreasing
LDL uptake (Fig. 5A) and thereby reducing the accumulation of
LDL-derived cholesterol in LE/Ly.
For such mutations, a sufficient activity of the proteins in the
appropriate organelles (LE/Ly) might be achieved either by making
more of the protein (i.e., by increasing transcription/translation) or
by increasing the efficiency of passage through the ER and Golgi
apparatus. A previous report described up-regulation of the NPC1
mRNA in response to cAMP, and a mechanism involving histone
acetylation was proposed (24). That study reported that treatment
with TSA increased NPC1 mRNA levels in steroidogenic Y-1 cells.
Recent studies have also documented induction of the 78 kDa glu-
cose regulated protein/binding immunoglobulin protein (GRP78/
BiP) and 70 kDa heat shock protein (HSP70) protein chaperones
by inhibition of class I HDACs (36, 37). This suggests that HDACi
could increase passage of mutant NPC1 out of the ER. Further
work will be required to establish the relative contributions of these
two mechanisms in increasing NPC1 expression in mutant cells.
Since the GM18453 cell line is homozygous for the NPC1I1061T
mutation, the HDACi treatment is effective on this variant of the
NPC1protein.TheGM03123cellscontainanNPC1I1061Talleleand
an NPC1P237Sallele. It is not clear whether the expression of the
P237S NPC1 protein is also increased by the HDACi treatments.
The activity of the benzamides, which are known to be inactive
against class II HDACs (35), suggests that class I HDACs are
responsible for the observed effects. Furthermore, the inactivity
of PCI34051 excludes HDAC8 as a potential target. Although the
effect of the thiophene benzamide suggests the closely related
HDAC1 or HDAC2 as a target, the observed activity and selec-
tivity of the compound is not sufficient to exclude HDAC3 as a
Fig. 3.
cells GM18445 in the presence of HDACi. NPC1 mutant human fibroblasts
GM18453 (A) and NPC2 mutant human fibroblasts GM18445 (B) were trea-
ted with CI-994, SAHA, LBH589, or TSA at various concentrations for 48 h
followed by fixing, staining with filipin, and imaging. DMSO was used as
a solvent control. Images were analyzed to obtain filipin LSO ratio, and data
were normalized to corresponding DMSO-treated cells. Data shown are
averages of three independent experiments totaling 60 images (five wells ×
four sites × three experiments). The dotted horizontal lines indicate mean
values for the solvent control; error bars represent SE.
Dose dependence for NPC1 mutant cells GM18453 and NPC2 mutant
Fig. 4.
and NPC1 mutant human fibroblasts GM03123 and GM18453 were treated
with LBH589, TSA, or SAHA. As a control, cells were treated with DMSO.
After 48 h, Western blot analysis was performed. The membrane was probed
with mouse monoclonal anti-human NPC1 antibody, and anti–α-actin was
used as a loading control.
Effect of HDACi on NPC1 expression. WT human fibroblast GM05659
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potential target. This conclusion is analogous to the situation with
the majority of the current HDACi approved or in clinical trials,
which broadly inhibit HDAC1-3 (38).
The finding that treatment of NPC1 mutant fibroblasts with
HDACi at submicromolar concentrations results in normalization
of cholesterol homeostasis may lead to treatment options for this
devastatingdisease, especially because many HDACi havealready
been studied in humans as drugs for a range of other diseases. The
most potent compound found in our studies, LBH589 (pan-
obinostat), is currently being used in several clinical trials (in-
cluding phase III trials) for various forms of cancer, including
glioblastomas, indicating that it can efficiently cross the blood–
brain barrier (39, 40). It is orally available, has good pharmacoki-
netic properties, and was shown to have fewer cardiac side effects
compared with many other HDACi (32, 41). Detailed pharmaco-
kinetic data showing half-lives of up to 17 h have been published
(42), and LBH589 has also been shown to be well-tolerated when
administered orally (43) or i.v. (44). Investigation of this and other
HDACi as therapeutics in animal models of NPC is, therefore,
a promising direction to explore in further studies.
Materials and Methods
Chemical Synthesis of HDAC Inhibitors. Synthesis and characterization of all
compounds are described in detail in SI Materials and Methods, including
schemes S1–S13.
Cell Culture.Human NPC1 fibroblasts GM03123 and GM18453, as well as NPC2
fibroblasts GM 18445 (Coriell Institute), were maintained in modified Eagle
medium (MEM) supplemented with 10% FBS.
Preparation of Lipoprotein-Deficient Serum and LDL. Lipoprotein-deficient
serum (LPDS) was obtained from FBS adjusted to a final density of 1.2 g/mL
(45). LDL was obtained from human plasma. LDL was labeled with 1,1’-
dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Invitro-
gen Corporation) as described elsewhere (46, 47).
Time- and Dose-Dependence Assay. The dose dependence of four HDACi
(CI-994, SAHA, LBH589, and TSA) was determined as a function of time after
4- to 72-h treatment of NPC1 mutant fibroblasts. Measurements were made
from four wells for each condition in each experiment; the experiment was
repeated two times. Images were acquired at 10× magnification on an
ImageXpressMicroautomatic fluorescence microscope at four sites per well
and analyzed to obtain the LSO compartment ratio (30). Additional details
are in SI Materials and Methods.
Fluorescence Microscopy. An ImageXpressMicroimagingsystem from Molecular
Devices equipped with a 300 W Xenon-arc lamp from Perkin-Elmer, a Nikon
10× Plan Fluor 0.3 NA objective, and a Photometrics CoolSnapHQ camera
from Roper Scientific was used to acquire images. Filipin images were ac-
quired using 377/50-nm excitation and 447/60-nm emission filters with a 415-
dichroic long-pass filter. DRAQ5 images were acquired using 628/40-nm ex-
citation and 692/40 nm-emission filters with a 669-dichroic long-pass filter.
Image Analysis. Images were analyzed to obtain the filipin LSO ratio using
MetaXpress Image Analysis Software as described previously (30).
Fig. 5.
GM03123 fibroblasts were treated with HDACi as described in Materials and
Methods. DiI intensity per cell area was measured after treatment with 10
μM CI-994, 0.04 μM LBH589, 10 μM SAHA, or 0.37 μM TSA. DMSO treatment
was used as a vehicle control, and WT GM05659 fibroblasts were used as
a reference. Data shown are from three independent experiments, and 20
images were acquired per experiment. Error bar represents SE, and P values
compared with DMSO control were <0.001 for all treatments (Student t
test). (B) SREBP2 processing. GM03123 cells were treated with 10 μM CI-994,
0.04 μM LBH589, 10 μM SAHA, or 0.37 μM TSA. As a control, cells were
treated with DMSO. After 18 h, Western blot analysis was performed. The
membrane was probed with mouse monoclonal anti-SREBP2 antibody. (C)
Characterization of HDACi. (A) DiI-LDL uptake. NPC1 mutant
Quantification of SREBP2. The ratio of cleaved SREBP2 to total SREBP2
(precursor + cleaved form) is plotted for treatments with HDACi, using
DMSO treatment as a control. Error bar represents SE, and P values com-
pared with DMSO control were <0.05 for all treatments (Student t test). (D)
ACAT assay. WT and NPC1 mutant GM05659 and GM03123 fibroblasts were
plated in growth medium with 10% FBS at 37 °C. ACAT assay was performed
as described in Materials and Methods. The nanomoles of cholesteryl-[14C]-
oleate per milligram cell protein were calculated. Data for HDACi-treated
cells were normalized to DMSO-treated controls and are presented as
mean ± SE. The LBH589-treated cells had a statistically significant (P < 0.05)
increase in esterification in the absence of the ACAT inhibitor (Student
Newman–Kuels multiple comparison). Two independent experiments were
conducted, and each experiment had three data points.
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DiI-LDL Uptake. Cells were plated in growth medium supplemented with 10%
FBS,andafter1d,mediumwas changed to5%LPDS.After 16–18h,cellswere
treated with HDACi in medium supplemented with 5% LPDS and 20 mM
Hepes. After 24 h of incubation, 5 μg/mL DiI-LDL were added to the cells and
incubated for additional 24 h in the presence of the HDACi. The cell area was
determined based on filipin labeling, and the average DiI intensity per cell
area was measured. Additional details are in SI Materials and Methods.
Western Blot. Western blot analysis was performed on WT and HDACi-
treated NPC1 mutant fibroblasts. Primary monoclonal anti-mouse NPC1,
Actin and GAPDH antibodies were from Invitrogen. Monoclonal antiSREBP2
antibody was from Santa Cruz. Secondary antibodies were either from
Pierce or Licor.
ACAT Assay. Cells were plated in Falcon 24 wells. The procedure described
previously (34, 48) was followed. The number of nanomoles of cholesteryl-
[14C]-oleate per milligram protein was determined for each well.
ACKNOWLEDGMENTS. We thank Harold Ralph for acquiring images on an
automated fluorescence microscope. We gratefully acknowledge financial
support from the Ara Parseghian Medical Research Foundation (to P.H., O.
W., and F.R.M.), a grant from the Charles Edison Fund (to P.H.), and Grant
R37-DK27083 from the National Institutes of Health (to F.R.M.).
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ApanobinostatPresurgery
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CHEMISTRY