翻页电子书制作,电子书制作,电子杂志制作

欢迎来到云展网,国内唯一的3D翻页电子书免费发布、阅读平台。上传PDF即可转换为翻页电子书!

13. Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease—翻页版预览

阅读,搜索本杂志文字内容 点击阅读
阅读云展网其他3D杂志 点击阅读
8787 上传于 2018-09-07 12:41:39

13. Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/51548679

Lanosterol induces mitochondrial uncoupling and protects dopaminergic
neurons from cell death in a model for Parkinson's disease

Article  in  Cell death and differentiation · August 2011 READS

DOI: 10.1038/cdd.2011.105 · Source: PubMed 144

CITATIONS Vernice Jackson-Lewis, PhD
Columbia University
28 150 PUBLICATIONS   16,978 CITATIONS   

10 authors, including: SEE PROFILE

Lynette Lim Guanghou Shui
King's College London Chinese Academy of Sciences
16 PUBLICATIONS   294 CITATIONS    248 PUBLICATIONS   6,932 CITATIONS   

SEE PROFILE SEE PROFILE

Loo Chin Wong
Duke-NUS Medical School
5 PUBLICATIONS   61 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:
Tuberculosis View project
Investigating the changes to sphingolipids and cholesteryl esters in Huntington's disease View project

All content following this page was uploaded by Vernice Jackson-Lewis, PhD on 21 May 2014.
The user has requested enhancement of the downloaded file.

Cell Death and Differentiation (2012) 19, 416–427

& 2012 Macmillan Publishers Limited All rights reserved 1350-9047/12

www.nature.com/cdd

Lanosterol induces mitochondrial uncoupling and
protects dopaminergic neurons from cell death in
a model for Parkinson’s disease

L Lim*,1, V Jackson-Lewis2, LC Wong3, GH Shui4, AXH Goh4, S Kesavapany4, AM Jenner4,7, M Fivaz3,5, S Przedborski2
and MR Wenk*,1,4,6

Parkinson’s disease (PD) is a neurodegenerative disorder marked by the selective degeneration of dopaminergic neurons in the
nigrostriatal pathway. Several lines of evidence indicate that mitochondrial dysfunction contributes to its etiology. Other studies
have suggested that alterations in sterol homeostasis correlate with increased risk for PD. Whether these observations are
functionally related is, however, unknown. In this study, we used a toxin-induced mouse model of PD and measured levels of
nine sterol intermediates. We found that lanosterol is significantly (B50%) and specifically reduced in the nigrostriatal regions of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, indicative of altered lanosterol metabolism during PD pathogenesis.
Remarkably, exogenous addition of lanosterol rescued dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP þ )-
induced cell death in culture. Furthermore, we observed a marked redistribution of lanosterol synthase from the endoplasmic
reticulum to mitochondria in dopaminergic neurons exposed to MPP þ , suggesting that lanosterol might exert its survival effect
by regulating mitochondrial function. Consistent with this model, we find that lanosterol induces mild depolarization of
mitochondria and promotes autophagy. Collectively, our results highlight a novel sterol-based neuroprotective mechanism with
direct relevance to PD.
Cell Death and Differentiation (2012) 19, 416–427; doi:10.1038/cdd.2011.105; published online 5 August 2011

Parkinson’s disease (PD) is a movement disorder marked by neurons via the dopamine transporter, inhibits mitochondrial

the selective degeneration of dopaminergic neurons in the complex I and eventually induces clinical symptoms

nigrostriatal pathway. About 5–10% of PD are genetically reminiscent of PD.
As MPTP/MPP þ toxicity emulate PD symptoms, it has
linked with mutations in genes such as leucine-rich repeat
been widely used in animal and cellular models to study
kinase 2 (LRRK2), alpha-synuclein, PTEN-induced putative
kinase 1 (PINK1), Parkin and DJ-1.1 These genetic PD cases, neuronal cell death and to screen for neuroprotective agents.

while rare, have provided insights into mechanisms of PD Of the neuroprotective metabolites identified, many are found

pathogenesis and many of which point toward mitochondrial in mitochondria, including L-carnitine, creatine and coenzyme
dysfunction.1–3 For example, PINK1, Parkin and DJ-1 control Q (CoQ)-10.6 Although their precise protective mechanisms

clearance of mitochondria by mitophagy in response to are still poorly understood, some evidence suggests that they
cellular stress.3 Mitochondrial defects are also seen in act as mitochondrial uncouplers.7 Consistent with this model,

idiopathic PD, whereby the catalytic activity of brain mito- uncoupling proteins (UCPs) are protective in the MPTP model
chondrial complex I is compromised.4 Finally, environmental of PD,8,9 and their expression is downregulated in DJ-1 knock-
out mice.10 Neuronal cell death induced by glutamate toxicity
toxins that affect complex I, such as 1-methyl-4-phenyl- can also be rescued by mitochondrial uncouplers.11
1,2,3,6-tetrahydropyridine (MPTP),5 can also induce
More recently, misregulation of sterol metabolism has
Parkinsonism. The active metabolite of MPTP, 1-methyl-4-
phenylpyridinium (MPP þ ), selectively enters dopaminergic also been implicated in PD. In clinical studies, elevation of

1Department of Biological Sciences, National University of Singapore, Singapore; 2Department of Neurology, Center for Motor Neuron Biology and Disease, Columbia
University, New York, USA; 3Program in Neuroscience and Neurobehavioral Disorders, DUKE-NUS Graduate Medical School, Singapore; 4Department of Biochemistry,
Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 5Department of Physiology, Yong Loo Lin School of Medicine, National University of
Singapore (NUS), Singapore and 6Swiss Tropical and Public Health Institute, University of Basel, Socinstrasse 57, Basel, Switzerland

*Corresponding authors: L Lim or MR Wenk, Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Life Sciences,
28 Medical Drive # 04-26D, Singapore 117456, Singapore. Tel: þ 65 6516 3624; Fax: þ 65 6777 3271; E-mail: g0601115@nus.edu.sg or markus_wenk@nuhs.edu.sg
7Current address: Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, New South Wales, Australia

Keywords: lanosterol; Parkinson’s disease; mitochondrial membrane potential; lipids; dopaminergic neruons

Abbreviations: ATP, adenosine triphosphate; AV, autophagosome vacuole; CCCP, m-chlorophenylhydrazone; CCD, charge-coupled device; cdk5, cyclin-dependent

kinase 5; CoQ, coenzyme Q/ Ubiquinone; DIV, days in vitro; DJ-1, (PARK7) Parkinson disease 7; ER, endoplasmic reticulum; GC–MS, gas chromatography–mass
spectrometry; GSK-3b, glycogen synthase kinase 3 beta; HPLC, high-pressure liquid chromatography; JC-1, 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazo-

locarbocyanine iodide; KDEL, ER retention sequence (lys-asp-glu-leu); LC3, microtubule-associated protein light chain 3; LDL-C, low-density lipoprotein cholesterol;
LRRK2, leucine-rich repeat kinase 2; LSS, lanosterol synthase; MPP þ , 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MRM,
multiple reaction monitoring; p35, cdk5 activator protein with 35 kDa; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PD, Parkinson’s disease; PINK1, PTEN-

induced putative kinase 1; SREBP2, sterol regulatory element-binding protein 2; TH, tyrosine hydroxylase; TOMM20, translocase of outer mitochondrial membrane 20;

TUJ1, neuron-specific class III beta tubulin; UCP, uncoupling protein; Dc, mitochondrial membrane potential

Received 01.3.11; revised 30.6.11; accepted 04.7.11; Edited by N Bazan; published online 05.8.11

Lanosterol protects dopaminergic neurons
L Lim et al

417

low-density lipoprotein cholesterol (LDL-C) in serum corre- [Fold difference]1.6 ventral midbrain
lates with higher prevalence of PD,12 whereas higher serum striatum
levels of total cholesterol are associated with a decreased risk squalene
of PD.13 Biochemical studies showed that alpha-synuclein, 1.4 lanosterol
the major component of Lewy bodies found in PD brains, lathosterol
binds to cholesterol with high affinity,14 and its aggregation is 1.2 7-dehydro
accelerated in presence of oxidized cholesterol.15 In a cholesterol
separate line of investigation, oxidative stress has been 1 desmosterol
shown to increase levels of lanosterol, a cholesterol pre- 7-β-OH
cursor, in mitochondria and several other intracellular 0.8 cholesterol
compartments of macrophages, suggesting that this sterol ***
metabolite may be part of a global cellular response to 7-keto
stress.16 Yet, how sterol metabolism is altered in the brains of 0.6 cholesterol
PD patients, or which sterol metabolites, if any, have an
impact on the survival of dopaminergic neurons is unknown. 0.4 24-OH
cholesterol
Our study investigated the role of sterol metabolism in the 0.2 cholesterol
MPTP/MPP þ model of PD. Our results provide evidence for
the specific role of lanosterol as a neuroprotective agent in 0
dopaminergic neurons. We also show that lanosterol induces
mild uncoupling of mitochondria and promotes autophagy, Figure 1 Lanosterol is the only sterol specifically depleted in affected brain
two events that have been previously linked to neuroprotec- areas of mice treated with MPTP. C576B6 mice were treated with the acute
tion in various models of PD. To our knowledge, our work schedule of MPTP injections. At 48 h after the last dose of MPTP, the ventral
provides the first link between sterol metabolism and midbrain and striatum were dissected, and lipids were extracted for analysis of sterol
mitochondrial function, and identifies lanosterol as a potential intermediates by GC–MS. Fold changes are plotted on the y axis, which represent
therapeutic agent for PD. the average levels from MPTP-treated animals (n ¼ 4) normalized to average levels
from saline-treated (control) animals (n ¼ 6) for each metabolite. Error bars
Results represent S.E.M. In both brain regions, the levels of lanosterol are reduced
significantly in MPTP-treated animals. ***Po0.001
Lanosterol levels are decreased in the striatum and
ventral midbrain of MPTP-injected mice. To determine if To determine if addition of lanosterol in culture affects other
sterol metabolism is altered in a rodent model of PD, we sterol intermediates, we analyzed an array of metabolites
measured levels of sterol metabolites in the striatum and (squalene, lanosterol, lathosterol, 7-dehydrocholesterol,
ventral midbrain of control and MPTP-treated mice by gas desmosterol, cholesterol, 7-b hydroxycholesterol, 7-keto-
chromatography-mass spectrometry (GC–MS). This acute cholesterol and 24-hydroxycholesterol) in extracts from
MPTP injection regime results in about 35% loss of ventral midbrain cultures by GC–MS. In cells treated with
dopaminergic neurons after 48 h.17 Of the nine metabolites lanosterol, we observed a 12-fold increase in lanosterol
analyzed, we found that lanosterol, the first cyclic sterol, was levels relative to control (no treatment, Figures 2d and e) and
reduced by B50% in affected areas (Figure 1). Owing to the no detectable effect on other sterol metabolites tested
specific and highly significant reduction in the levels of (Figure 2e), suggesting that cells have a high capacity to
lanosterol in the affected areas of MPTP-treated animals, we accumulate lanosterol, consistent with previous findings in
reasoned that lanosterol might be important for dopaminergic macrophages.16 This is not the case for cholesterol, as we did
neuronal survival. not detect any changes in cholesterol levels on exogenous
addition of cholesterol, perhaps because neuronal cholesterol
Lanosterol protects dopaminergic neurons from MPP þ homeostasis is tightly regulated.18 In addition to the metabo-
-induced cell death. We asked whether exogenous addition lites indicated above, we also tested the impact of lanosterol
of lanosterol protects dopaminergic neurons against MPP þ - and cholesterol addition on levels of ubiquinones, isoprenoid-
induced cell death. Primary postnatal neuron cultures from the derived electron carriers in mitochondria, and found no
ventral midbrain were treated with MPP þ , and the survival of significant differences (Figures 3a and b).
dopaminergic neurons was determined in the absence or
presence of exogenously added lanosterol. In all, 48% of In these experiments, postnatal dopaminergic neurons
dopaminergic neurons survived on treatment with MPP þ were grown in direct contact with a glia feeder. It is therefore
under these culture conditions (Figures 2a and c). possible that neuroprotection is indirectly mediated by a
Co-incubation of MPP þ with phosphatidylcholine (PC, modification of astrocyte physiology. To address whether
vehicle control) or cholesterol did not improve survival of lanosterol addition leads to a specific increase in lanosterol
dopaminergic neurons (Figures 2b and c). In contrast, co- concentration in neurons, we repeated these experiments
treatment of the cultures with MPP þ and lanosterol increased using hippocampal neurons cultured according to Banker’s
survival of dopaminergic neurons to 76% (Figures 2b and c). method,19 which allows physical separation of neurons and
Thus, lanosterol, but not cholesterol or PC, rescues astrocytes. We found that lanosterol levels increased in both
dopaminergic neurons from MPP þ -induced cell death. neurons and astrocytes to a similar extent on treatment with
exogenous lanosterol (data not shown). Although we cannot
rule out the possible contribution of astrocytes in lanosterol-
mediated neuroprotection, our results indicate that lanosterol
levels are significantly elevated in neurons, arguing for a direct
role of lanosterol in promoting survival of dopaminergic
neurons.

Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

418

a 10μM MPP+ c 100

Control (no treatment) 10μM MPP+ & 80
5μM Lanosterol % of DA neurons ***
b 10μM MPP+ & [treatment / control]
60
5μM PC
40

20

0

- + + + + MPP+
- - + - - PC
- - - + - Lanosterol
- - - - + Cholesterol

10μM MPP+ &
5μM Cholesterol

d e 18 5μM PC

Non-treated 16 *** 5μM cholesterol
(control)
5μM PC 14 5μM lanosterol
5μM
cholesterol 12
5μM
lanosterol 10
cells treated with lipids
8
sterol metabolites
cells with no treatment (ctrl) 6

[Fold difference] 4

internal standard 2
(ion m/z 493,
100ng deuterated-β-sitosterol) 0
squalelanneostelarothlost7e-crdohelohleydstrdeoer-osml osterocl7h-okeletsoct-ehroolel sterol
lanosterol (ion m/z 393)

Figure 2 Lanosterol rescues dopaminergic neurons in MPP þ -treated ventral midbrain cultures. Fluorescence images of primary ventral midbrain cultures (DIV7) stained

with anti-TH, a marker for dopaminergic neurons. (a) Control cells (no treatment) and cells treated with 10 mM MPP þ ; (b) co-treated cells for 24 h with 10 mM MPP þ and

5 mM of phosphatidylcholine (PC, left panel), lanosterol (middle panel) or cholesterol (right panel). Scale bars in (a) and (b) represent 200 mm. (c) Plot of fold changes of each

treatment relative to control. y axis shows the average percentage of TH þ neurons from each treatment condition divided by the average percentage of TH þ neurons in
control. Error bars represent S.E.M. from four to five independent experiments. ***Po0.001 between lanosterol/MPP þ and MPP þ . (d) GC–MS profiles of lanosterol
(m/z 393, light grey) and the internal standard (deuterated b-sitosterol, m/z 493, black) in ventral midbrain cultures incubated for 24 h with various lipid treatments. (e) Cellular

levels of sterol intermediates following various treatments normalized to control (no treatment). Fold changes plotted in y axis represent the levels of each metabolite in each
treatment condition normalized to control. Error bars represent S.E.M. from three independent experiments. ***Po0.001 between lanosterol treatment and control

To explore the mechanisms underlying lanosterol-mediated SREBP2 levels, which was not rescued by lanosterol
neuroprotection, we next investigated the effects of MPP þ addition (Figure 4a, top panel). We also checked for levels
and lanosterol on various signaling pathways previously of cdk5 activator protein with 35 kDa (p35), the activator of
implicated in cellular metabolism and neurodegeneration. cyclin-dependent kinase 5 (cdk5), because the genetic
First, we examined the expression level of sterol response ablation of p35/cdk5 confers neuroprotection in the MPTP
element-binding protein (SREBP2, Figure 4a), a transcription model.21 Although we observed a decrease in p35 levels in
factor reported to exacerbate neuronal degeneration.20 MPP þ -treated cells, addition of lanosterol did not restore
We found that MPP þ induced a modest increase in p35 expression to control levels (Figure 4a, second panel

Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

419

a b

Geranyl-PP 0.05 Q8 Q9 Q10 total

Squalene + Tyrosine/ Relative coenzyme Q levels 0.04
paraquinone

Ubiquinone 0.03
0.02

Lanosterol 0.01

Sterol 0 PC Cholesterol Lanosterol
biosynthesis
Control

Figure 3 Measurements of ubiquinone on addition of lipids. (a) Simplified pathway to show the cross-talk between sterol and ubiquinone biosynthesis in mammalian cells.
Both pathways use isoprenyl units as precursors. (b) Plot of ubiquinone/CoQ levels normalized to total cholesterol levels in neuronal cultures from various treatment
conditions. Error bars represent S.E.M. over four independent experiments

from top). Consistent with previous findings implicating Figure 5a, bottom last panel), with concomitant reduction in
glycogen synthase kinase 3 beta (Gsk-3b) (Ser9) phosphor- the overlap of LSS with KDEL (R2 ¼ 0.69±0.02, Figure 5a,
ylation in PD pathogenesis,22 we found increased phospho- second panel from the bottom). Translocation of LSS from the
Gsk-3b in MPP þ -treated cells. However, this increase was ER to mitochondria was also observed in dopaminergic
not affected by the addition of lanosterol (Figure 4a, third neurons co-treated with MPP þ and lanosterol (Figure 5b).
panel from top). MPP þ did not, however, affect LSS localization in non-
dopaminergic neurons (Figure 5b), consistent with the inability
Finally, we examined the levels of lanosterol synthase of these cells to take up MPP þ . These data indicate that a
(LSS), the enzyme that catalyzes cyclization of oxidosqua- significant fraction of LSS redistributes from the ER to
lene, the rate limiting step in lanosterol synthesis.23 The levels mitochondria in dopaminergic neurons exposed to MPP þ ,
of LSS did not markedly change on MPP þ addition suggesting that LSS (and its product lanosterol) may have a
(Figure 4a, fourth panel from top). Interestingly, however, role in regulating mitochondrial function.
we observed a different localization pattern of LSS in MPP þ
-treated dopaminergic neurons. LSS immunostaining Lanosterol uncouples mitochondria in dopaminergic
appeared much more punctate after MPP þ treatment neurons. An important aspect of mitochondrial physiology
(Figure 4b), suggesting drug-induced redistribution of LSS can be assessed by measuring the mitochondrial membrane
to a different intracellular compartment. We thus proceeded to potential (Dc), which is the electrical and chemical gradient
examine the subcellular localization of LSS in control and that drives protons across the inner membrane during
MPP þ -treated dopaminergic neurons. electron transport and oxidative phosphorylation. This
process is never completely ‘coupled’, allowing a fraction of
Redistribution of LSS in dopaminergic neurons on electrons and protons to be transported without concurrent
MPP þ insult. LSS is a membrane-associated enzyme, production of adenosine triphosphate (ATP). We first
which is targeted to the cytoplasmic leaflet of the examined the impact of lanosterol on Dc in hippocampal
endoplasmic reticulum (ER).24 Biochemical analysis of neurons using the ratiometric voltage-sensitive fluorescent
purified fractions from mouse liver showed that the bulk of dye, 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolocarbo-
LSS is indeed associated with the microsomal/ER fractions cyanine iodide (JC-1). Using time-lapse, multi-positioning
and that minor amounts are found in purified mitochondria imaging to capture about 3–4 fields in 30-s intervals, we
(Figure 4c). measured changes of Dc on lipids addition by the ratiometric
analysis of JC-1 red to green emission. As expected,
Consistent with our biochemical fractionation data, we treatment of neurons with 200 nM m-chlorophenylhydrazone
found that LSS colocalized with an ER marker (ER retention (CCCP), a known uncoupler of oxidative phosphorylation,
sequence (lys-asp-glu-leu) (KDEL)) in both dopaminergic and induced an immediate and sharp reduction of the
non-dopaminergic neurons (Figure 5a). Extensive overlap mitochondrial membrane potential (B25% within 30 s;
between LSS and the ER marker was observed for both Figure 6a). Under these same experimental conditions,
dopaminergic and non-dopaminergic neurons (R2 ¼ 0.83± exogenous addition of lanosterol reduced the membrane
0.01 and 0.84±0.01, respectively). Less overlap was found potential by B20% over 15 min, whereas PC and cholesterol
between LSS and translocase of outer mitochondrial mem- had no significant effect (Figure 6b).
brane 20 (TOMM20) (a mitochondrial marker) for both
dopaminergic and non-dopaminergic neurons (R2 ¼ 0.70± We repeated these experiments in ventral midbrain cultures
0.02 and 0.69±0.02, respectively, Figure 5a, second panel (Figures 6c and d). As these cultures were grown at a lower
from the top). This pattern changed noticeably in dopaminer- density than the hippocampal cultures, we needed to capture
gic neurons treated with MPP þ . The colocalization of LSS 10–15 fields in time-lapse multi-positioning imaging at lower
with TOMM20 was markedly increased (R2 ¼ 0.856±0.01,

Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

420

magnification to gain enough statistical power. As a conse- As MPP þ uptake depends on ATP levels,25 it is possible
quence, this limited time intervals to 2 min. We observed a that the addition of lanosterol causes mitochondrial depolar-
B20% of reduction in membrane potential on lanosterol ization, and subsequently ATP depletion. This would, in turn,
addition (Figures 6c and d), consistent with the results inhibit MPP þ uptake in the mitochondria. To determine if
obtained in hippocampal neurons. lanosterol alters ATP levels, we measured levels of ATP in

a SREBP2 b LSS CONTROL TH
TH
MPP+ - + + + + - - - p(s21/9) dopaminergic
PC - - + - - + - - Gsk- MPP+ (24 HOURS)
non-dopaminergic
Lanosterol - - - + - - + - p35 3α / β LSS LSS
Cholesterol - - - - + - - + dopaminergic
TH
100 non-dopaminergic
70
55

37

25
55

α
β
37
100
70

70
55

c 5μg 10μg

WL M C ER WL M C ER

150

100
70

55 Lanosterol synthase
(78 kDa)

150

100
70

Calnexin-
55 ER marker (90 kDa)

37

VDAC mito marker
25 (31k Da)

Figure 4 Analyses of SREBP2, Gsk-3b, p35/cdk5 and LSS in ventral midbrain treated with lipids and MPP þ . (a) Immunoblot analyses of several factors previously linked
to neuroprotection in PD and/or MPTP treatment. (b) Immunofluorescence images of ventral midbrain neurons stained with LSS and TH treated with or without MPP þ . LSS
appears more punctate in TH þ neurons on MPP þ treatment. (c) Subcellular fractionation of liver tissue. M, mitochondrial, ER, microsome/ER, C, cytosolic, WL, fractions
were purified from whole liver. Immunoblot of calnexin (ER maker) and VDAC/porin (mitochondrial marker) were used to assess the purity of each fraction. LSS showed a
clean single band at the expected weight of 78 kDa and is enriched in the ER fraction. A small amount of LSS is also detected in the mitochondrial fraction

Figure 5 LSS is redistributed from ER to mitochondria in dopaminergic neurons on addition of MPP þ . (a) Confocal images of ventral midbrain neurons stained with
TH (white), LSS (green), either KDEL (red, first and third panels from the top) or TOMM20 (red, second and fourth panels from the top) of ventral midbrain neurons in control
and MPP þ treatment. White boxes show dopaminergic neurons as determined by TH þ staining. Right panels show pixel intensity correlation plots of LSS with either KDEL
or TOMM20 in dopaminergic neurons. Scale bar represents 10 mm. (b) Average R2 values (a measure of colocalization) of two classes of neurons in control and MPP þ
-treated conditions. In control, R2 values from co-staining of KDEL-LSS, n ¼ 26 and n ¼ 27, TOMM20-LSS, n ¼ 20 and n ¼ 21, were assessed for dopaminergic and non-
dopaminergic neurons respectively. In MPP þ -treated cells, R2 values from co-staining of KDEL-LSS, n ¼ 20 and n ¼ 20, for TOMM20-LSS, n ¼ 18 and n ¼ 17, were
assessed for dopaminergic and non-dopaminergic neurons respectively. In MPP þ /lanosterol co-treated cells, R2 values from co-staining of KDEL-LSS, n ¼ 18 and n ¼ 23,
for TOMM20-LSS, n ¼ 19 and n ¼ 18, were assessed for dopaminergic and non-dopaminergic neurons respectively. ****Po0.00001

Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

421

neurons treated with PC, cholesterol or lanosterol. We did not that lanosterol is unlikely to inhibit MPP þ uptake. Thus, one
see any significant changes in the levels of ATP across all mechanism by which lanosterol could mediate neuroprotec-
treatment conditions (Supplementary Figure S1), indicating tion is through the uncoupling of mitochondria.

a KDEL LSS-KDEL-DAPI intensity in LSS channel (green) dopaminergic neuron

TH LSS 4000

CONTROL (NO TREATMENT) R2 = 0.89

3000

2000

1000

TH LSS TOMM20 LSS-TOMM20-DAPI intensity in LSS channel (green) 0 1000 2000 3000 4000
KDEL intensity in KDEL channel (red)

4000

R2 = 0.72

3000

2000

1000

TH LSS LSS-KDEL-DAPI intensity in LSS channel (green) 0 1000 2000 3000 4000
TH LSS intensity in TOMM20 channel (red)
MPP+ (24 HOURS)
4000

R2 = 0.76

3000

2000

1000

TOMM20 LSS-TOMM20-DAPI intensity in LSS channel (green) 0 1000 2000 3000 4000
intensity in KDEL channel (red)

4000

R2 = 0.89

3000

2000

1000

0 1000 2000 3000 4000
intensity in TOMM20 channel (red)

b TOMM20-LSS KDEL-LSS ****
****
1.00 ****
0.90 ****
co-localization coefficient (R2)0.80
neurToHn+0.70
neuortohner0.60
neurToHn+
neuortohner
neurToHn+
neuortohner

Control MPP+ Lanosterol & MPP+

Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

422 a 200nM of CCCP added b Lipid added (time = 240s)

c 1.1Change in membrane potential 1.1
1.0 (Δf/f0 [JC-1 red/green])
0.9 1.0
Change in membrane potential
(Δf/f0 [JC-1 red/green]) 0.9

0.8 0.8
0.7
400 800 0.7 5μM PC (n = 60)
0 Time (seconds) 0.6 5μM Cholesterol (n = 60)
5μM Lanosterol (n = 63)
0
400 800 1200
Time (seconds)

Time = 0 min Time = 40 mins post-stain with anti-TH

control

+Lanosterol

d 1.2 Lipid added (time = 6 minutes)

1.1
Change in membrane potential
(Δf/f0 [JC-1 red/green]) 1.0

0.9

0.8

0.7 Control (n=14)
0.6 PC (n=13)
Cholesterol (n=12)
0 Lanosterol (n=13)

5 10 15 20 25 30 35 40
Time (minutes)

Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

423

Lanosterol increases autophagy in dopaminergic On the other hand, evidence for a role of aberrant sterol
neurons. Previous studies have shown that the loss of metabolism in PD is rather controversial. For example, lower
mitochondrial membrane potential can initiate the autophagic levels of serum LDL-C are considered a risk factor for PD,12,32
degradation of damaged mitochondria.26,27 Gene linked to but it is unclear how serum LDL-C relates to levels of
familial forms of PD, such as PINK1 and Parkin, are thought cholesterol in the brain. Statins, which lower serum cholester-
to regulate this process, and PD-associated Parkin mutations ol levels by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme
cause a decrease in mitophagy in mammalian cell lines.28 A reductase, have been used to treat PD, but there are many
We thus asked if lanosterol mediates autophagosome unresolved questions regarding their benefits (see review in
formation in dopaminergic neurons. Using microtubule- Becker and Meier33).
associated protein light chain 3 (LC3) as a marker for
autophagy, we quantified both the size and number of In this study, we measured a selection of sterol metabolites
autophagic vacuoles (AVs) in primary ventral midbrain in the ventral midbrain and striatum of MPTP-treated mice,
neurons on MPP þ /lanosterol treatment. Similar to other two brain regions characterized by cell death and/or axonal
studies,29 we found that addition of MPP þ in primary loss in both the MPTP model as well as in PD. Our results
dopaminergic neurons increased the number of AVs by show a highly selective reduction of lanosterol levels in these
about 2.5-fold (Figure 7). There is also about a 75% increase affected areas (Figure 1) and point to an alteration of
in the average size of AVs with MPP þ treatment. lanosterol metabolism in MPTP-treated mice. We cannot
Remarkably, when neurons were exposed to lanosterol rule out the possibility that lanosterol is oxidized or metabo-
alone, we observed a similar increase in AV size and lized to di-hydrolanosterol or other products in MPTP-treated
number. Co-treatment of MPP þ and lanosterol led to an animals because we are unable to measure these oxidized
additive effect on autophagy, with significant increases in metabolites.
both the number and size of AVs, compared with MPP þ or
lanosterol alone (Figure 7). Consistent with a role of lanosterol in PD pathogenesis,
we observed an improved survival of MPP þ -treated dopa-
Next, we addressed whether lanosterol has a specific effect minergic neurons on exogenous addition of lanosterol
on axonal mitophagy. In PD, axons of dopaminergic neurons (Figure 2). We also demonstrated that LSS relocalized from
progressively degenerate and ‘die back’, in a process that the ER to mitochondria in dopaminergic neurons on MPP þ
may be accelerated by mitochondrial dysfunction and involve treatment (Figures 4b and 5), suggesting an increase in
mitophagy. For this, we grew hippocampal neurons in lanosterol synthesis in mitochondria. Interestingly, recent
microfluidic chambers to segregate axons from neuronal cell lipidomic analysis of macrophages during stimulation with
bodies and dendrites.30 Mitochondria were visualized by Lipid A (a condition that leads to oxidative bursts) showed
expression of the fluorescent reporter MitoRed and neurons a pronounced increase in lanosterol levels in several
were immunostained for endogenous LC3. We found a small intracellular compartments, including mitochondria,16 impli-
but significant increase in colocalization of MitoRed with LC3 cating that modulation of lanosterol metabolism may be part of
(Figure 8) on lanosterol treatment, suggesting an increase in a global cellular response to stress. If upregulation of
axonal mitophagy. Taken together, these results suggest that lanosterol synthesis is part of a cellular defense mechanism,
the protective effects of lanosterol are mediated by mitochon- it is not clear why lanosterol levels drop in response to
dria uncoupling, and subsequent clearance of damaged MPTP treatment. In this regard, it is interesting to note that
mitochondria. two recent papers reported lowered lanosterol levels in the
serum of patients with Alzheimer’s disease,34 and in
Discussion fibroblasts challenged by virus infection.35 One possible
explanation for these seemingly contradicting results is that
The etiology of PD implicates several factors, including distinct types of stress differentially impact lanosterol meta-
mitochondrial dysfunction and misregulation of sterol meta- bolism. Perhaps, in some cases, the substantial decrease in
bolism. Evidence implicating impaired mitochondrial function lanosterol levels cannot be compensated by upregulation of
in PD is substantial. This evidence is based on (1) the lanosterol synthesis as part of the cell’s protective response.
identification of rare PD-associated mutations in genes that Alternatively, we cannot exclude the possibility that transloca-
affect mitochondrial function such as the putative kinase, tion of LSS to mitochondria in response to MPP þ is an
PINK1 (PARK6), the E3 ligase Parkin (PARK2) and DJ-1 epiphenomenon, which is not indicative of a cellular response
(PARK7); (2) similarities between PD and clinical symptoms to stress.
that arise on exposure to the neurotoxin MPTP, a complex I
inhibitor; and (3) a significant decrease in complex I/II activity Our results, however, show that exogenous addition of
in the platelets of patients with PD.31 lanosterol leads to mild uncoupling of mitochondria in
dopaminergic and glutamatergic neurons, with no detectable
impact on ATP levels (Figure 6 and Supplementary Figure

Figure 6 Lanosterol induces uncoupling of the mitochondria in dopaminergic neurons. Live imaging of JC-1 loaded neuronal cultures. (a) Control plot showing changes in
JC-1 red to green ratio, Df/f0, versus time in hippocampal neurons treated with CCCP, a known uncoupler, n ¼ 20. (b) Plot of Df/f0 versus time in hippocampal neurons treated
with different lipids, n460 for each condition from three independent experiments. (c) Images of control and lanosterol-treated ventral midbrain cultures at the start of the
experiment (time ¼ 0 min, left panels) and the end of the experiment (time ¼ 40 min, middle panels). Right panels represent posteriori staining of TH for identification of
dopaminergic neurons. White boxes represent magnified TH þ neurons. Scale bar represents 100 mm in main figure and 10 mm in magnified box. (d) Plot of Df/f0 versus time
for dopaminergic neurons, n410 for each condition from three independent experiments. In both types of cultures, lanosterol induces about 20% decrease in mitochondrial
membrane potential

Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

424

a control lanosterol MPP+ Lanosterol & MPP+

AV identified LC3 TH

b 70 number of AV *** 70
avg size of AV (pixel area) 60
60 50
50 *** 40 AV average pixel area/cell
40
Number of AV/ cell ***

30 30

20 20

10 10

00
- + - - - + - - PC

- - + - - - + - Cholesterol

- - - + - - - + Lanosterol
- - - - + + + + MPP+

Figure 7 Lanosterol and MPP þ increase the number of AVs in dopaminergic neurons. (a) Confocal images of ventral midbrain neurons stained with TH (top panels), LC3
(middle panels) and AV quantification (ImageJ software output, bottom panels) with various treatment conditions indicated on the top. Scale bar represents 10 mm.
(b) Graphical plot showing the average number of AV identified per TH þ cells and the average size of AV in pixel area during treatment with various lipids and MPP þ
co-treatment. For each condition, n440 TH þ cells from three independent experiments. Error bars represent S.E.M. ***Po0.001

S1). The mitochondria uncoupling and protective effects of associated Parkin mutant proteins fail to translocate.27,38
lanosterol are strikingly similar to those observed with low Together, these data point to a role of mitochondrial
dose of the uncoupler, FCCP, which improves cellular survival uncoupling and autophagy in PD pathogenesis. In line with
in ischemic preconditioning but has no significant impact on this model, our results reveal that lanosterol induces
ATP levels.36 In the context of PD, the mitochondrial mitochondrial uncoupling (Figure 6) and promotes autophagy
uncoupling effect of lanosterol has important implications. (Figures 7 and 8).
For example, Parkin is recruited to mitochondria via PINK1 on
membrane depolarization,26 and regulates the clearance of Although mitochondrial uncoupling has been shown to be
damaged mitochondria by mitophagy in mammalian cell neuroprotective in various models, including MPTP-induced
lines.37 In addition, the translocation of Parkin to mitochondria neurodegeneration,7–9 the mechanisms involved are still
has etiological significance, as a number of disease- unclear. Some studies have shown that uncoupling reduces
superoxide species, offering an explanation for improved
Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

425

a control lanosterol b *
*
MitoRed LC3 4
% of Mitochondrial 3
with LC3 2
1
0

control
RLCaahpnoalosemtsyterrcPioollnC

Figure 8 Lanosterol increases mitophagy in axons. (a) Confocal images of hippocamal axons stained with LC3 (top panels, green) and electroporated with MitoRed

(middle panels, red). Bottom panels show of LC3 and MitoRed. White arrows represent-colocalization of the two channels. Scale bar represents 20 mm. (b) Graphical plot of

average percentage of mitochondria with LC3-positive stains. Error bars represent S.E.M. For each condition, n440 axons were assessed from three independent
experiments. *Po0.05

neuronal survival in the MPTP model, because oxidative Lanosterol, cholesterol and PC were dissolved in chloroform/methanol (1 : 1).
stress is thought to be the primary cause of cell death.8,9 In Cholesterol or lanosterol was mixed in equimolar proportion with PC and dried by
other studies, transient mitochondrial uncoupling is neuropro- vacuum in a Speedvac (Thermosavant, Waltham, MA, USA). The lanosterol/PC or
tective in glutamate-induced neurotoxicity, as it prevents cholesterol/PC mixture was resuspended in culture medium on the day of treatment
uptake of calcium from the cytosol to mitochondria.11 Finally, a to make a 0.5 mM stock liposome. Each type of stock liposome was used in the
recent study showed that DJ-1, a gene involved in early onset neuronal cultures within a day of preparation.
PD, regulates the expression of two UCPs (UCP4 and UCP5)
and controls oxidative stress in mitochondria of dopaminergic Lipid extraction. For tissues and cells, extraction of lipids was performed using
neurons in the substantia nigra.10 Although these studies Bligh and Dyer method.39 Briefly, cells were washed 3–5 times with phosphate-
cited above have identified different modulators by which a buffered saline (PBS), scraped in 400 ml ice-cold methanol and transferred to a
cell/neuron alters mitochondrial membrane potential, they are 1.5-ml Eppendorf tube. Chloroform (200 ml) was added and samples were vortex for
in good agreement with our findings, whereby uncoupling 1 min. Next, 300 ml of 1 M KCL was added, and the homogenates were
mechanism proves to be a central regulator of cellular microcentrifuged at 14 000 r.p.m. for 5 min at 4 1C to separate phases. The lower
response to stress. organic phase was carefully transferred to a new Eppendorf tube. The aqueous
phase was re-extracted twice with 300 ml chloroform. All organic phases were
In conclusion, we report that in addition to its role as a pooled and dried under vacuum using a Speedvac and stored at À80 1C until
precursor for cholesterol biosynthesis, lanosterol acts as a derivatization and subsequent GC–MS analysis.
survival factor for dopaminergic neurons. Furthermore, our
findings point to an unexpected role of this sterol metabolite in Ventral midbrain (B20–25 mg) and striatum (B15–20 mg) were dissected and
regulating mitochondrial function and autophagy, and bring tissues were homogenized using a Dounce homogenizer in 600 ml ice-cold
sterol metabolism to the forefront of neurobiological disease. chloroform/methanol (1 : 2). Another 300 ml chloroform was added to the
homogenate followed by the addition of 450 ml of 1 M KCL. The homogenates
Materials and Methods were microcentrifuged at 14 000 r.p.m. for 5 min at 4 1C. The lower organic phase
PD animal model: MPTP injections. All procedures performed in rodents was carefully transferred to a new Eppendorf tube, and the aqueous phase was re-
were in accordance with IACUC guidelines. MPTP injections were performed extracted twice with 300 ml chloroform. All organic phases were pooled and dried
according to previously published methods, following the acute schedule.17 Briefly, under vacuum in a Speedvac. Dried samples were then stored at À80 1C until
C57B6 mice were given four i.p. doses of either 18 mg/kg of MPTP (Sigma-Aldrich, derivatization and subsequent analyses.
St. Louis, MO, USA) or saline (control) every 2 h. Mice were decapitated 48 h after
the last dose, and the ventral midbrain and striatum were dissected and snap-frozen Sample preparation for GC–MS. Briefly, dried lipid extracts were
for subsequent lipid extraction and GC–MS analysis. Previously published data resuspended in chloroform/methanol (1 : 1) to a concentration of 0.1 mg tissue/ml
using the same protocol showed that at this timepoint, about 35% of dopaminergic solvent. A 20-ml sample of lipid extract was removed and completely dried in a glass
neurons have degenerated.17 vial. For each sample, we added a mixture of heavy isotopes: 40 ng of
7a-hydroxycholesterol-d7, 40 ng of 7b-hydroxycholesterol-d7, 40 ng of 26(27)-
Lipid standards and liposomes. Lanosterol, cholesterol, 1,2-dimyristoyl- hydroxycholesterol-d5, 80 ng of 7-ketocholesterol-d7, 0.2 mg of 5a-cholestane,
sn-glycero-3-phosphocholine (DMPC or PC) and desmosterol-d6 (all of highest 0.2 mg of desmosterol-d6, 0.2 mg of lathosterol-d4, 0.2 mg of campesterol-d7 and
purity, 499%) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 0.2 mg of b-sitosterol-d7 in 25 ml of ethanol. Standards and sample mixtures were
Oxysterol standards a-cholestane, 7a-hydroxycholesterol, 7b-hydroxycholesterol, dried under a stream of N2 before adding the derivatizing agent (15 ml acetonitrile
7-dehydrocholesterol, 25-hydroxycholesterol and 7-ketocholesterol were and 15 ml BSTFA þ TMCS; Pierce Thermoscientific, Waltham, MA, USA). The
obtained from Sigma (St. Louis, MO, USA). 7a-Hydroxycholesterol-d7, derivatized samples were analyzed with an Agilent 5975 inert XL mass selective
7b-hydroxycholesterol-d7, b-sitosterol-d7, campesterol-d3, lathosterol-d4 and detector (Santa Clara, CA, USA). Selective ion monitoring was performed using the
7-ketocholesterol-d7 were purchased from CDN Isotopes (Quebec, Canada). electron ionization mode at 70 eV (with the ion source maintained at 230 1C and the
27-Hydoxycholesterol-d5, 24-hydroxycholesterol and 24-hydroxycholesterol-d7 quadrupole at 150 1C) to monitor one target ion. Two qualifier ions were selected for
were purchased from Medical Isotopes (Pelham, AL, USA). Deuterated the mass spectrum of each compound to optimize for sensitivity and specificity.
standards obtained were of 495% purity.
Ventral midbrain cultures. Ventral midbrains from 20 postnatal day 0 to day
2 rodents were dissected and digested in papain solution and plated on a glia layer.
Cells were cultured in serum-free neurobasal/B27 medium (Invitrogen, Carlsbad,
CA, USA) supplemented with superoxide dismutase 1 (5 mg/ml), apo-transferrin
(95 mg/ml) and insulin (21 mg/ml) (all from Sigma). In contrast to Rayport et al.,40 our

Cell Death and Differentiation

Lanosterol protects dopaminergic neurons
L Lim et al

426

culture medium contained no serum because the cultures were subjected to lipid automated stage and built in autofocusing system (PFS, Nikon, Tokyo, Japan) and
addition in experimental conditions. One hour after plating the cells, 10 ng/ml of glial- driven by Metamorph 7.6 (Universal Imaging, Ypsilanti, MI, USA). JC-1 is excited at
derived neurotrophic factor (Millipore, Billerica, MA, USA) was added. To inhibit glia 488-nm, and its fluorescence emission was collected at 530±10 nm (green) and
growth, a solution of 16.5 mg/ml uridine and 6.7 mg/ml 5-fluorodeoxyuridine was 590±17 nm (red), corresponding to peak fluorescence from the monomer and
added 1 day after plating. Cells were then cultured for 7 days (days in vitro (DIV)7) aggregate signals, respectively. Mitochondrial membrane potential was measured
before treatment with MPP þ and various types of liposome. by taking the red to green emission ratio.

Hippocampal neuron cultures. Hippocampal neurons from rats E18.5 For ventral midbrain cultures (DIV7), cells were seeded at 50 cells/mm2 in a
embryos were cultured in neurobasal/B27 as described.19 Briefly, neurons were glass-bottom labtek well. Cells were loaded with 1 mg/ml JC-1 (Invitrogen) in culture
plated on a wax-dotted coverslip coated with 1 mg/ml of poly-D-lysine (Sigma). Two medium, and were incubated for 30 min at 37 1C, washed twice with HBSS and
hours after plating, when neurons had attached, the coverslips were flipped into a imaged in conditioned culture medium. Multi-position time-lapse imaging of 10–15
six-well plate containing a glia feeder layer. PC, cholesterol or lanosterol liposome randomly chosen fields was performed at 2-min interval over 40 min. At the end of
(5 mm each) was added after DIV7. the experiment, ventral midbrain cells were fixed on stage for 20 min with 4% PFA
and stained for TH with a secondary antibody coupled to Alexafluro-568 (red) and
Western and antibodies. For western blots, cultured cells were washed DAPI. The retrospective staining of TH allowed for the identification of dopaminergic
three times with 1 Â PBS. Cells were lysed in 100 ml of RIPA buffer (50 mM Tris- neurons, which were the only ones included in the analyses. Hippocampal cultures
HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl and 1 mM EDTA), were plated at a higher density (300 cells/mm2), and multi-position time-lapse
supplemented with a cocktail of protease inhibitors (Complete Mini-EDTA inhibitors, imaging of 3–4 fields was performed at 30-s intervals for 20 min.
Roche Diagnostics, Indianapolis, IN, USA). In all, 20 mg of cell protein lysate was
loaded in each well of a 10% polyacrylamide gel containing 0.1% SDS. After For both types of cultures, the intensity of the JC-1 red-to-green ratio was
electrophoresis, proteins were transferred to nitrocellulose membrane and probed measured in each frame, and the change in mitochondrial membrane potential was
with the following antibodies: (i) rabbit anti-p35 (C19; 1 : 1000, Santa Cruz plotted as Df/f0, where f0 is the average JC-1 red-to-green ratio over the first 10
Biotechnology, Santa Cruz, CA, USA), (ii) rabbit anti-LSS (1 : 1000, AVIA, Systems frames before treatment. Decay curves were fitted to a mono-exponential function,
Biology, San Diego, CA, USA), (iii) rabbit anti-tyrosine hydroxylase (TH) (1 : 20 000, y ¼ x0eÀt/t, using IGOR Pro 6.1 (WaveMetrics Inc., Lake Oswego, OR, USA).
Covance, Princeton, NJ, USA), (iv) rabbit anti-SREBP2 (1 : 1000, Abcam,
Cambridge, MA, USA), (v) rabbit anti-calnexin (1 : 2000, Abcam), (vi) rabbit anti- Measurement of CoQ. For each condition, 3 to 4 million hippocampal
VDAC/porin (1 : 2000, Abcam) and (vii) rabbit anti-pGSK-3a/b (ser21/9) 1 : 1000, neurons (DIV7) were treated with various lipids and incubated for 24 h. Cells were
Cell Signaling, Danvers, MA, USA). Peroxidase-conjugated anti-rabbit or anti- washed two times with cold PBS and scraped in 0.5 ml of cold PBS. Cells were
mouse secondary antibodies (1 : 10 000) were purchased from Bio-Rad (Hercules, centrifuged at 1000 Â g to pellet the cells, and resuspended in 100 ml fresh PBS.
CA, USA). Immunoblots were visualized with enhance chemiluminescence reagent Next, 750 ml of hexane/ethanol (5 : 2 v/v) was added, and samples were vigorously
from Pierce Thermoscientific. vortexed for 1 min. To extract CoQ, 400 ml of the organic phase was collected and
completely dried under a stream of N2, followed by LC–MS analysis.
Fluorescence microscopy and quantification of dopaminergic
neuronal survival. Ventral midbrain cultures plated on 12-mm coverslips were An Agilent HPLC 1200 system coupled with an Applied Biosystems 3200 QTrap
treated for 24 h with 10 mM MPP þ with or without 5 mM PC, 5 mM cholesterol or mass spectrometer (Foster City, CA, USA) was used for measuring CoQ8, Q9, and
5 mM lanosterol liposome. Cells were washed three times with PBS, then fixed with Q10 and free cholesterol. Chromatographic separation was carried out using an
4% paraformaldehyde for 20 min, followed by permeabilization and blocking in 5% Agilent Zorbax Eclipse XDB-C18 column (i.d. 4.6 Â 150 mm). High-pressure liquid
fetal bovine serum in 0.1% TritonX-100 for 30 min. Cells were then stained with anti- chromatography (HPLC) conditions were as the following: mobile phase:
TH (secondary: Alexa-fluor 488, green) and neuron-specific class III beta tubulin chloroform/methanol 1 : 1 (v/v); flow rate: 0.5 ml/min; column temperature: 30 1C;
(TUJ1) (secondary: Alexa-fluor 555, red). Anti-mouse and anti-rabbit Alexa-fluor 488 injection volume: 10 ml. The LC–MS instrument was operated in positive
and 555 (1 : 1000) were purchase from Molecular Probes (Eugene, OR, USA)/ atmospheric pressure chemical ionization mode with a vaporizer temperature of
Invitrogen. TUJ1-positive and TH-positive cells were counted with an Olympus 500 1C and a corona current of 3 mA. CoQ6 was used as an internal standard and
fluorescence microscope (Tokyo, Japan) with FITC and TRITC filter sets. Every monitored with an multiple reaction monitoring (MRM) transition of 591.4197.0.
neuron on the 12-mm coverslip was counted. The percentage of dopaminergic MRM transitions of 727.5197.0, 795.5197.0 and 863.6197.0 were set up for analysis
neurons in each group was determined by the number of TH þ /TUJ1 þ cells. of CoQ8, Q9 and Q10, respectively.
Typically, in each control coverslip, there were 2000–3000 TUJ1 þ cells, of which
400–1200 were TH þ . For each treatment, we assessed 4–6 coverslips per Quantification of AVs. Ventral midbrain cultures plated on 12-mm coverslips
independent experiment. The averages of 4–5 independent experiments are shown were treated for 24 h with 10 mM MPP þ with or without 5 mM PC, 5 mM cholesterol
in the figures. or 5 mM lanosterol. Cells were washed three times with PBS, then fixed with 4%
paraformaldehyde for 20 min and permeabilized with 100 mg/ml of digitonin for
Confocal microscopy and colocalization studies. Ventral midbrain 10 min. Following permeabilization, cells were washed three times with PBS and
cultures in control and MPP þ -treated cells were stained with rabbit anti-LSS stained with anti-TH (secondary: Alexa-fluor 488, green) and anti-LC3 (1 : 100,
(1 : 100), monoclonal mouse anti-TOMM20 (1 : 500) (Abcam) or monoclonal mouse mouse monoclonal, MBL cat. no.: 152–3A, secondary: Alexa-fluor 555, red). To
anti-KDEL (1 : 500) (Abcam), and sheep anti-TH (1 : 500) (Abcam). The secondary quantify AV in dopaminergic neurons, TH þ cell soma were imaged with a laser-
antibodies, goat anti-mouse Alexa-fluor 555, goat anti-rabbit Alexa-fluor 488 and scanning confocal microscope (LSM510, Carl Zeiss) with excitation and emission
donkey anti-sheep Alexa-fluor 633, were obtained from Invitrogen. Cells were filters meeting the secondary Alexa-fluor antibody dye specifications. Images were
imaged with a laser-scanning confocal microscope (LSM510, Carl Zeiss, taken using a 63X objective with the same laser power and gain. The 12-bit images
Oberkochen, Germany) with excitation and emission filters meeting the were quantified using ImageJ (analyze particle drop-in, National Institute of Health,
secondary Alexa-fluor antibody dye specifications. Images were taken using a Bethesda, MD, USA). For each image, detected LC3 puncta were intensity thresholded
63X objective. To quantify colocalization, we plotted the pixel intensities of LSS (o1000) and gated for size (o15 pixel). For each condition, 40–60 TH þ cells were
versus KDEL or TOMM20 from regions of interest (ROIs) drawn around single assessed from three independent experiments.
neurons (either TH-positive or -negative), and calculated the linear regression
coefficient, R2, for 16–22 individual ROI/neuron. As a positive control for our method of evaluating AV, mouse embryonic
fibroblast grown in serum or serum deprived were stained with LC3 and quantified
Live-cell confocal imaging and measurement of mitochondrial according to the same parameters. As expected and shown in supplementary data
membrane potential. Mitochondrial membrane potential was measured in (Supplementary Figure S2), there is approximately a 25-fold increase in AV on
live neurons using JC-1. All live-imaging experiments were conducted in cell serum starvation.
medium, (37 1C and 5% CO2) with a spinning disk confocal microscope, equipped
with a Cool SNAP HQ2 CCD camera (Photometrics, Tucson, AZ, USA), a fully Quantification of mitophagy in axons. E18.5 hippocampal neurons
were electroporated using the Amaxa poration system (Lonza, Basel, Switzerland)
Cell Death and Differentiation with MitoRed construct (Clonetech, Mountain View, CA, USA, cat. No.: PT-3633-5).
Cells were then plated in microfluidic chambers (Xona Microfluidics, Temecula, CA,
USA), which allow for physical separation of axons and cell somas. At DIV7,

Lanosterol protects dopaminergic neurons
L Lim et al

427

neurons were treated with 5 mM PC, 5 mM cholesterol, 5 mM lanosterol or 200 nM of 16. Andreyev AY, Fahy E, Guan Z, Kelly S, Li X, McDonald JG et al. Subcellular organelle
rapamycin (positive control) for 24 h. Cells were then fixed with 4% lipidomics in TLR-4-activated macrophages. J Lipid Res 2010; 51: 2785–2797.
paraformaldehyde for 20 min and permeabilized with 100 mg/ml of digitonin for
10 min. Following permeabilization, cells were washed three times with PBS, and 17. Jackson-Lewis V, Jakowec M, Burke RE, Przedborski S. Time course and morphology of
stained with anti-LC3 (secondary: Alexa-fluor 488, green). To quantify mitophagy, dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-
the percentage of MitoRed and LC3-positive mitochondria were plotted as a tetrahydropyridine. Neurodegeneration 1995; 4: 257–269.
percentage to total MitoRed-positive mitochondria using the Metamorph 7.6
colocalization drop-in. For each condition, images of 40–50 axons were taken from 18. Vance JE, Hayashi H, Karten B. Cholesterol homeostasis in neurons and glial cells. Semin
three independent experiments. Cell Dev Biol 2005; 16: 193–212.

Statistical analyses. Error bars represent S.E.M. For fold changes in sterol 19. Kaech S, Banker G. Culturing hippocampal neurons. Nat Protoc 2006; 1: 2406–2415.
intermediates, P-values were calculated with two-tailed Mann–Whitney U-test. For 20. Fernandez A, Llacuna L, Fernandez-Checa JC, Colell A. Mitochondrial cholesterol loading
in vitro assessment of dopaminergic neuron survival, CoQ levels, ATP levels, AV
number and size, percentage of mitophagy, P-values were calculated with exacerbates amyloid beta peptide-induced inflammation and neurotoxicity. J Neurosci
two-tailed Student’s t-test. 2009; 29: 6394–6405.
21. Neystat M, Rzhetskaya M, Oo TF, Kholodilov N, Yarygina O, Wilson A et al. Expression of
Conflict of Interest cyclin-dependent kinase 5 and its activator p35 in models of induced apoptotic death in
neurons of the substantia nigra in vivo. J Neurochem 2001; 77: 1611–1625.
The authors declare no conflict of interest. 22. Garcia-Gorostiaga I, Sanchez-Juan P, Mateo I, Rodriguez-Rodriguez E, Sanchez-
Quintana C, del Olmo SC et al. Glycogen synthase kinase-3 beta and tau genes interact in
Acknowledgements. We thank Thilo Hagen for providing us with an aliquot of Parkinson’s and Alzheimer’s diseases. Ann Neurol 2009; 65: 759–761 author reply
CCCP. This study was supported by the National University of Singapore (NUS), 761-752.
Department of Biological Sciences fellowship, the Singapore Biomedical Research 23. Mori M, Sawashita J, Higuchi K. Functional polymorphisms of the LSS and Fdft1 genes in
Council (BMRC) Grant ID 08/1/21/19/558, the Singapore National Research laboratory rats. Exp Anim 2007; 56: 93–101.
Foundation under CRP Award No. 2007-04 and the SystemsX.ch RTD project LipidX. 24. Yamamoto S, Lin K, Bloch K. Some properties of the microsomal 2,3-oxidosqualene sterol
cyclase. Proc Natl Acad Sci U S A 1969; 63: 110–117.
1. Lesage S, Brice A. Parkinson’s disease: from monogenic forms to genetic susceptibility 25. Ramsay RR, Singer TP. Energy-dependent uptake of N-methyl-4-phenylpyridinium, the
factors. Hum Mol Genet 2009; 18 (R1): R48–R59. neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, by mitochondria.
J Biologic Chem 1986; 261: 7585–7587.
2. Dauer W, Przedborski S. Parkinson’s disease: mechanisms and models. Neuron 2003; 39: 26. Narendra D, Tanaka A, Suen DF, Youle RJ. Parkin is recruited selectively to impaired
889–909. mitochondria and promotes their autophagy. J Cell Biol 2008; 183: 795–803.
27. Matsuda N, Sato S, Shiba K, Okatsu K, Saisho K, Gautier CA et al. PINK1 stabilized by
3. Cookson MR. DJ-1, PINK1, and their effects on mitochondrial pathways. Mov Disord 2010; mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent
25(Suppl 1): S44–S48. Parkin for mitophagy. J Cell Biol 2010; 189: 211–221.
28. Geisler S, Holmstrom KM, Treis A, Skujat D, Weber SS, Fiesel FC et al. The PINK1/Parkin-
4. Keeney PM, Xie J, Capaldi RA, Bennett Jr JP. Parkinson’s disease brain mitochondrial mediated mitophagy is compromised by PD-associated mutations. Autophagy 2010; 6:
complex I has oxidatively damaged subunits and is functionally impaired and 871–878.
misassembled. J Neurosci 2006; 26: 5256–5264. 29. Cherra 3rd SJ, Kulich SM, Uechi G, Balasubramani M, Mountzouris J, Day BW et al.
Regulation of the autophagy protein LC3 by phosphorylation. J Cell Biol 2010; 190: 533–539.
5. Liu Y, Yang H. Environmental toxins and alpha-synuclein in Parkinson’s disease. Mol 30. Park JW, Kim HJ, Byun JH, Ryu HR, Jeon NL. Novel microfluidic platform for culturing
Neurobiol 2005; 31: 273–282. neurons: culturing and biochemical analysis of neuronal components. Biotechnol J 2009; 4:
1573–1577.
6. Virmani A, Gaetani F, Binienda Z. Effects of metabolic modifiers such as carnitines, 31. Haas RH, Nasirian F, Nakano K, Ward D, Pay M, Hill R et al. Low platelet mitochondrial
coenzyme Q10, and PUFAs against different forms of neurotoxic insults: complex I and complex II/III activity in early untreated Parkinson’s disease. Ann Neurol
metabolic inhibitors, MPTP, and methamphetamine. Ann NY Acad Sci 2005; 1053: 1995; 37: 714–722.
183–191. 32. Huang X, Abbott RD, Petrovitch H, Mailman RB, Ross GW. Low LDL cholesterol and
increased risk of Parkinson’s disease: prospective results from Honolulu-Asia Aging Study.
7. Horvath TL, Diano S, Leranth C, Garcia-Segura LM, Cowley MA, Shanabrough M et al. Mov Disord 2008; 23: 1013–1018.
Coenzyme Q induces nigral mitochondrial uncoupling and prevents dopamine cell loss in a 33. Becker C, Meier CR. Statins and the risk of Parkinson disease: an update on the
primate model of Parkinson’s disease. Endocrinology 2003; 144: 2757–2760. controversy. Expert Opin Drug Saf 2009; 8: 261–271.
34. Kolsch H, Heun R, Jessen F, Popp J, Hentschel F, Maier W et al. Alterations of cholesterol
8. Andrews ZB, Horvath B, Barnstable CJ, Elsworth J, Yang L, Beal MF et al. Uncoupling precursor levels in Alzheimer’s disease. Biochimica et biophysica acta 2011; 1801: 945–950.
protein-2 is critical for nigral dopamine cell survival in a mouse model of Parkinson’s 35. Blanc M, Hsieh WY, Robertson KA, Watterson S, Shui G, Lacaze P et al. Host defense
disease. J Neurosci 2005; 25: 184–191. against viral infection involves interferon mediated down-regulation of sterol biosynthesis.
PLoS Biol 2011; 9: e1000598.
9. Conti B, Sugama S, Lucero J, Winsky-Sommerer R, Wirz SA, Maher P et al. Uncoupling 36. Brennan JP, Southworth R, Medina RA, Davidson SM, Duchen MR, Shattock MJ.
protein 2 protects dopaminergic neurons from acute 1,2,3,6-methyl-phenyl- Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent
tetrahydropyridine toxicity. J Neurochem 2005; 93: 493–501. cardioprotection independent of KATP channel activation. Cardiovasc Res 2006; 72: 313–321.
37. Geisler S, Holmstrom KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ et al. PINK1/Parkin-
10. Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT et al. mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol 2010; 12:
Oxidant stress evoked by pace making in dopaminergic neurons is attenuated by 119–131.
DJ-1. Nature 2010; 468: 696–700. 38. Okatsu K, Saisho K, Shimanuki M, Nakada K, Shitara H, Sou YS et al. p62/SQSTM1
cooperates with Parkin for perinuclear clustering of depolarized mitochondria. Genes Cells
11. Stout AK, Raphael HM, Kanterewicz BI, Klann E, Reynolds IJ. Glutamate-induced neuron 2010; 15: 887–900.
death requires mitochondrial calcium uptake. Nat Neurosci 1998; 1: 366–373. 39. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem
Physiol 1959; 37: 911–917.
12. Huang X, Chen H, Miller WC, Mailman RB, Woodard JL, Chen PC et al. Lower low-density 40. Rayport S, Sulzer D, Shi WX, Sawasdikosol S, Monaco J, Batson D et al. Identified
lipoprotein cholesterol levels are associated with Parkinson’s disease. Mov Disord 2007; postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.
22: 377–381. J Neurosci 1992; 12: 4264–4280.

13. de Lau LM, Koudstaal PJ, Hofman A, Breteler MM. Serum cholesterol levels and the risk of This work is licensed under the Creative Commons
Parkinson’s disease. Am J Epidemiol 2006; 164: 998–1002.
Attribution-NonCommercial-No Derivative Works 3.0
14. Broersen K, Brink D, Fraser G, Goedert M, Davletov B. Alpha-synuclein adopts an alpha- Unported License. To view a copy of this license, visit http://
helical conformation in the presence of polyunsaturated fatty acids to hinder Micelle creativecommons.org/licenses/by-nc-nd/3.0
formation. Biochemistry 2006; 45: 15610–15616.

15. Bosco DA, Fowler DM, Zhang Q, Nieva J, Powers ET, Wentworth Jr P et al. Elevated levels
of oxidized cholesterol metabolites in Lewy body disease brains accelerate alpha-synuclein
fibrilization. Nat Chem Biol 2006; 2: 249–253.

Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)

Cell Death and Differentiation

View publication stats

点击阅读翻页书版本