Liver X receptor � (LXR�): A link between�-sitosterol and amyotrophic lateralsclerosis–Parkinson’s dementiaHyun-Jin Kim*, Xiaotang Fan*, Chiara Gabbi*, Konstantin Yakimchuk*, Paolo Parini†, Margaret Warner*,and Jan-Åke Gustafsson*‡

Departments of *Biosciences and Nutrition and †Laboratory Medicine, Karolinska Institutet, Novum, SE-14186 Stockholm, Sweden

Contributed by Jan-Åke Gustafsson, December 13, 2007 (sent for review November 11, 2007)

Administration of �-sitosterol (42 mg/kg per day) for 3 weeks to8-month-old male LXR��/� mice resulted in the death of motorneurons in the lumbar region of the spinal cord and loss of tyrosinehydroxylase-positive dopaminergic neurons in the substantianigra. In mice at 5 months of age, �-sitosterol had no observedtoxicity but at 16 months of age, it caused severe paralysis andsymptoms typical of dopaminergic dysfunction in LXR��/� mice.WT mice were not affected by these doses of �-sitosterol. In5-month-old mice, levels of the intestinal transporters, ABCG5/8and Niemann-Pick C1 Like 1, were not affected by loss of liver Xreceptor (LXR) � and/or treatment with �-sitosterol nor were therechanges in plasma levels of cholesterol or �-sitosterol. In 8-month-old LXR��/� mice there was activation of microglia in the substan-tia nigra pars reticulata and aggregates of ubiquitin and TDP-43 inthe cytoplasm of large motor neurons in the lumbar spinal cord.Brain cholesterol concentrations were higher in LXR��/� than intheir WT counterparts, and treatment with �-sitosterol reducedbrain cholesterol in both WT and LXR��/� mice. In LXR��/� micebut not in WT mice levels of 24-hydrocholesterol were increasedupon �-sitosterol treatment. These data indicate that multiplemechanisms are involved in the sensitivity of LXR��/� mice to�-sitosterol. These include activation of microglia, accumulation ofprotein aggregates in the cytoplasm of large motor neurons, anddepletion of brain cholesterol.

central nervous system � cholesterol � microglia � neurodegenerativedisease � nuclear receptor

The factors responsible for the amyotrophic lateral sclerosis(ALS)-parkinsonism dementia complex (PDC), found com-

monly in Guam, have been intensively investigated and recentlyreviewed (1). Studies have focused on the role of neurotoxicityof sterol glucosides found in the seeds of Cycas circinalis that arepart of the diet in Guam (2) and on the neurotoxin �-methyl-amino-alanine found in cyanobacteria (3). The spinal cords ofALS patients are characterized by pathological accumulation ofsphingomyelin, ceramides, and cholesterol esters (4), which arethought to sensitize motor neurons to programmed cell deathand cytoplasmic accumulation of aggregates of ubiquitin andtransactivation response DNA-binding protein (TDP-43) (5, 6).

The role of phytosterols in the motor neuron degeneration inALS is still being investigated (1). The major dietary phytoster-ols are �-sitosterol, campesterol, and stigmasterol. Phytosterolsand their derivatives are known as ligands for liver X receptor(LXR) � and LXR� (7, 8). They are structurally similar tocholesterol and are abundant in plant oils, nuts, seeds, andfat-rich food such as avocados. Phytosterols and cholesterolcompete with each other for uptake in the jejunum via Niemann-Pick C1 Like 1 (NPC1L1) transporter (9). Cholesterol is trans-ported to the endoplasmic reticulum, where it is esterified by theaction of acyl-CoA:cholesterol O-acyltransferase 2 (Acat2) forincorporation into chylomicrons (10). However, plant sterols arepoor Acat2 substrates and are transported back to the luminal

membrane to be resecreted into the lumen of the intestine by theABCG transporters (10). ABC transporters are regulated byLXR� and LXR�. One member of the family, ABCG5, has beenimplicated as an etiological agent in motor neuron disease.Mutations in ABCG5 and ABCG8 have been found in sitoste-rolemic patients, but those subjects do not develop motor neurondisease (11–16). Similarly, mice in which ABCG5 and ABCG8have been inactivated develop sitosterolemia but not motorneuron disease (17).

Because of their size and high metabolic activity large motorneurons are very sensitive to oxidative stress and excitotoxinssuch as glutamate, both of which are known causes of motorneuron disease (18, 19). The immune system also plays a role inthe disease, and dendritic cells and activated microglia/macrophages have been detected in ALS spinal cord tissue (20).Wilson and Shaw (21) have demonstrated that mice fed with theglucuronide of �-sitosterol develop ALS, but glutamate trans-porters were not involved. Thus at least in �-sitosterol-inducedALS, glutamate toxicity is not causative.

LXR� and LXR� are members of the nuclear receptorsupergene family of ligand-activated transcription factors (22,23). Their endogenous ligands are oxysterols (24, 25). Studies onLXR� and LXR� knockout mice have revealed that LXR� butnot LXR� plays an important role in cholesterol homeostasis,whereas LXR� has key functions in the immune system and CNS(26–30). LXR� mRNA is widely expressed in the fetal rat brain,but in the postnatal and adult brains it is more discretelylocalized (31).

When both LXRs are inactivated (LXR���/� mice), severalsevere abnormalities have been observed in the brain (30). Incontrast, when only LXR� is inactivated (LXR��/� mice), by 6months of age, there is motor dysfunction that progresses withage into hind limb paralysis (32). Concomitant with worseningmotor dysfunction with age is a degeneration of the large�-motor neurons in the ventral horn of the spinal cord similar towhat is seen in ALS.

Oxysterols, known natural ligands of LXRs, are formed fromcholesterol either by cytochrome P450 enzymes or autoxidation.P450 subfamily members that produce oxysterols include cho-lesterol 7�-hydroxylase (CYP7A), cholesterol 27-hydroxylase(CYP27), and cholesterol 24-hydroxylase (CYP46). CYP46,which is found almost exclusively in brain, produces 24-hydroxycholesterol (33). Some cholesterol is transported fromthe brain to the cerebrospinal f luid via an ApoE-dependent

Author contributions: M.W. and J.-Å.G. designed research; H.-J.K., X.F., C.G., K.Y., P.P., andM.W. performed research; C.G. and P.P. contributed new reagents/analytic tools; H.-J.K.,M.W., and J.-Å.G. analyzed data; and H.-J.K., M.W., and J.-Å.G. wrote the paper.

Conflict of interest statement: J.-Å.G. is a shareholder and consultant for KaroBio AB.

‡To whom correspondence should be addressed. E-mail: jan-ake.gustafsson@mednut.ki.se.

This article contains supporting information online at www.pnas.org/cgi/content/full/0711599105/DC1.

© 2008 by The National Academy of Sciences of the USA

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mechanism. However, CYP24 is the rate-limiting enzyme re-sponsible for elimination of a majority of cholesterol from thebrain in the form of 24-hydroxycholesterol. Oxysterols can easilytraverse lipophilic membranes and, as ligands for LXRs, theyregulate a broad range of biological effects, including cholesterolhomeostasis, lipid metabolism, and the immune system.

Given the motor-neuron phenotype of the LXR��/� mice, wechose to use these mice to investigate toxicity of �-sitosterol, aknown motor neuron toxin (4, 8, 34, 35). We found that�-sitosterol is indeed more neurotoxic to LXR��/� mice than totheir WT counterparts, hastening the onset of ALS-like diseaseand causing degeneration of motor neurons and dopaminergicneurons in the substantia nigra.

ResultsImpaired Motor Coordination After �-Sitosterol Administration inMale LXR��/� Mice. Before �-sitosterol administration, mice of allages (3, 5, 8, and 16 months) used in these experiments weretested separately for their performance on a rotor rod. All3-month-old LXR��/�mice performed as well as WT mice, and3 weeks of �-sitosterol treatment did not change their perfor-

mance. Fore-limb muscle strength measured with the wirehanging test was normal in vehicle-treated and �-sitosterol-treated LXR��/� mice (data not shown).

At 5 and 8 months of age, the onset of the motor disability inLXR��/� mice as judged by the rotor rod test became apparent,but fore-limb muscle strength measured on the wire hanging testwas normal (data not shown). After 3 weeks of �-sitosteroladministration, retention time on a rotor rod in 5-month-old WTmice (89 � 2 s) was not different from �-sitosterol-treated mice(87 � 7 s). LXR��/� mice stayed on the rotor rod for 69 � 21 s,a duration not significantly different from that of WT mice (P �0.09). However, �-sitosterol treatment significantly (P � 0.009)reduced staying time on the rotor rod to 40 � 7 s (Fig. 1B).

At 8 months of age, retention time on a rotor rod for WT miceafter 3 weeks of �-sitosterol administration (88 � 2 s) was notdifferent from vehicle-treated mice (86 � 6 s). LXR��/� micestayed on the rotor rod for 62 � 9 s, a significantly shorter time(P � 0.01) than WT mice. �-Sitosterol administration furthersignificantly (P � 0.03) reduced staying time on the rotor rod to26 � 33 s (Fig. 1 A). As expected on the basis of our previousstudies, there was significant motor disability in 16-month-oldLXR��/� mice before �-sitosterol treatment, and after 3 weeksof �-sitosterol treatment, disability became more pronounced(Fig. 1C). The values on the rotor rod were 22 � 14 s forvehicle-treated LXR��/� mice and 8 � 6 s for �-sitosterol-treated LXR��/� mice (P � 0.01). At this age, WT miceperformed as well as 3-month-old mice. In addition, two of five�-sitosterol-treated LXR��/� mice lay on their sides in theircages, paralyzed, and rotated rapidly in circles parallel to thefloor when held by their tails. This type of behavior was neverobserved in WT or vehicle-treated LXR��/� mice. For ethicalreasons, treatments of 16-month-old LXR��/� mice with�-sitosterol were discontinued.

Tyrosine Hydroxylase (TH)-Positive Dopaminergic Neurons in 8-Month-Old Male Mice. At 8 months of age in WT mice, neurons werehealthy with TH-positive cell bodies and processes (Fig. 2 A andE). The loss of LXR� led to an overall weaker staining (Fig. 2C and G). TH-positive dopaminergic neuronal cell bodies in�-sitosterol-treated LXR��/� mice were shrunken with reducednumber of projections (Fig. 2 D and H). �-Sitosterol had noremarkable effects on neuronal morphology in the substantianigra of WT mice (Fig. 2 B and F).

Loss of Motor Neurons in Spinal Cords in 8-Month-Old Male LXR��/�

Mice. Nissl staining revealed that in LXR��/� mice, administra-tion of �-sitosterol resulted in significant (P � 0.002) loss of the

Fig. 1. Retention time of male mice on the rod. (A) 8-month-old mice. (B)5-month-old mice. (C) 16-month-old mice. n � 5 for each group. *, P � 0.05;

**, P � 0.01; ***, P � 0.001; Student’s t test.

A B C D

E F G H

Fig. 2. Representative TH immunohistochemical staining of coronal sections from the substantia nigra of 8-month-old male mice. Ten WT and 10 LXR��/� mice(8 months of age) were divided into four groups: group 1 (A and E), WT mice given olive oil only; group 2 (B and F), WT mice given 42 mg/kg �-sitosterol dissolvedin olive oil per day; group 3 (C and G), LXR��/� mice given olive oil only; and group 4 (D and H), LXR��/� mice given 42 mg/kg �-sitosterol dissolved in olive oilper day. Olive oil and �-sitosterol were given as a daily oral gavage for 3 weeks. In groups 1, 2, and 3, the shape of the neurons is clearly delineated by TH staining.Neuronal cell bodies in �-sitosterol-treated LXR��/� mice (group 4) were shrunken and less well stained for TH and there were fewer projections. E–H are close-upviews of the areas indicated in A–D. *, substantia nigra pars reticulata; #, substantia nigra pars compacta. (Scale bars: A–D, 100 �m; E–H, 10 �m.)

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large motor neurons in the lateroventral horns of L1 segmentsof the spinal cord (Fig. 3). The large motor neurons in WT micewere not affected by �-sitosterol. Data are expressed as numberof large motor neurons per section. The values were: group 1, 6 �2; group 2, 5 � 1; group 3, 3 � 1; group 4, 2 � 0 (group 1 vs. group3, P � 0.04; group 3 vs. group 4, P � 0.002; n � 5 for each group).This loss of large motor neurons observed with Nissl staining wasconfirmed by staining for the neuron-specific protein, microtu-bule-associated protein 2B (MAP2B) [see supporting informa-tion (SI) Fig. 8].

Intestinal Levels of ABC Transporters and NPC1L1 and Serum Levels of�-Sitosterol and Cholesterol in 5-Month-Old Male Mice. In LXR��/�

mice, levels of ABC transporters were normal (Fig. 4A), andlevels of �-sitosterol and cholesterol were similar to those in WTmice (Fig. 4 B and C). It appears that LXR�, which is expressedin LXR��/� mice, is sufficient for maintaining normal expres-sion of ABC transporters. Because levels of �-sitosterol were notelevated in LXR��/� mice, the neurotoxicity seen when thesemice were treated with �-sitosterol is likely caused by an increasein sensitivity of neurons to this neurotoxin.

CD11b-Positive Microglia in the Substantia Nigra of 8-Month-Old MaleMice. TH-positive neurons in the substantia nigra were detectedwith FITC-conjugated secondary antibody (Fig. 5 A–D). Activatedmicroglia are characterized by hyperramifications that stain posi-

tively for CD11b (Fig. 5); the number of activated microglia washigher in the substantia nigra pars reticulata of LXR��/� than inWT mice. �-Sitosterol had no remarkable effects on the number ofactivated microglia in the substantia nigra.

Immunohistochemistry of Cellular Inclusions, Ubiquitin and TDP-43, inLarge Motor Neurons of 8-Month-Old Male Mice. TDP-43 is alow-molecular-weight neurofilament mRNA-binding proteinthat is colocalized with ubiquitin in inclusions in motor neuronsin ALS (36). In the large motor neurons of vehicle-treatedLXR��/� mice, compact rounded ubiquitin-positive andTDP43-positive inclusions were observed in neuronal cytoplasm(Fig. 6 B and D). In the large motor neurons of vehicle- or�-sitosterol-treated WT mice, ubiquitin immunoreactivity wasweak and homogeneously distributed throughout the cytoplasm(Fig. 6 A and C). There were so very few large motor neurons leftin �-sitosterol-treated LXR��/� mice that it was difficult to findgood examples of inclusion bodies.

Concentration of Cholesterol and 24-Hydroxycholesterol in Brains of8-Month-Old Male Mice. The cholesterol contents in the brains ofLXR��/� mice were significantly higher than those in WT mice

Fig. 4. Relative expression of ABCG5, ABCG8, and NPC1L1 mRNA in theintestines of 5-month-old male mice. (A) Values of vehicle-treated WT micewere taken as 1; all other values are expressed relative to vehicle-treated WTmice. After 3 weeks of �-sitosterol administration, there were no measurablechanges in the expression of ABCG5, ABCG8, or NPC1 L1 in the intestines. (Band C) In the serum of these mice, the ratio of �-sitosterol/cholesterol (B) andconcentration of cholesterol (C) were unaffected by �-sitosterol treatment orloss of LXR� (n � 5 for each group).

Fig. 5. CD11b-positive microglia in the substantia nigra of 8-month-old malemice. (A and E) Group 1. (B) Group 2. (C and F) Group 3. (D) Group 4. (A–D)TH-positive dopaminergic neurons in the substantia nigra were detected byFITC-conjugated secondary antibody. Staining of the same section with CD11bantibody revealed activation of microglia defined as cells with hyperramifiedappearance in the substantia nigra pars reticulata. (E) Microglia (vehicle-treated WT mice) showed dense cell bodies without ramification. Treatmentwith �-sitosterol had no effect on morphology of microglia in WT mice (datanot shown). However, there were numerous activated microglia in vehicle-treated-LXR��/� mice (C and F), but the number was not increased by �-sitosterol treatment (data not shown). (E and F) Close-up views of the areasindicated in A and C, respectively. (Scale bars: A–D, 100 �m; E and F, 20 �m.)

A B

C D

E

Fig. 3. Large motor neurons in L1 segments of spinal cord of 8-month-oldmale mice. (A–D) Representative images of Nissl staining of spinal cord: group1 (A), group 2 (B), group 3 (C), and group 4 (D). (Scale bars: 50 �m.) (E) Thenumber of Nissl-stained large motor neurons in both ventral horns of each L1segment was evaluated. There were fewer large motor neurons in �-sitoster-ol-treated LXR��/� mice than in vehicle-treated counterparts (n � 5 for eachgroup; bar: mean � SD). Arrows indicate large motor neurons. The data areexpressed as the number of large motor neurons per section. The values are:group 1, 6 � 2; group 2, 5 � 1; group 3, 3 � 1; group 4, 2 � 0; group 1 vs. group3, *, P � 0.04; group 3 vs. group 4, **, P � 0.002; Student’s t test.

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(Fig. 7A). Administration of �-sitosterol caused a decrease incholesterol content in the brains of both LXR��/� and WT mice.Data are expressed as micrograms of cholesterol per milligramof brain tissue. The values were: group 1, 20.9 � 5.2; group 2,13.8 � 2.3; group 3, 27.3 � 9.6; group 4, 13.7 � 3.3; group 1 vs.group 3, P � 0.02; group 3 vs. group 4, P � 0.004; n � 5–14 foreach group.

The major route of excretion of cholesterol from the brain is

via its conversion to the more soluble metabolite, 24-hydroxycholesterol, a reaction that is catalyzed by CYP46 (33).There was a significant increase in the concentration of 24-hydroxycholesterol in response to �-sitosterol treatment in thebrains of LXR��/� but not in WT mice (Fig. 7B). Data areexpressed as nanograms of 24-hydroxycholesterol per milligramof brain tissue. The values were: group 1, 34.26 � 15.50; group2, 30.54 � 14.45; group 3, 32.79 � 17.02; group 4, 47.15 � 7.54(group 3 vs. group 4, P � 0.04; n � 5 for each group).

DiscussionIn the present study we found that �-sitosterol was not toxic to3-month-old LXR��/� mice but did alter motor function and wastoxic to dopaminergic neurons in the substantia nigra inLXR��/� mice at 5, 8, and 16 months of age. Because the onsetof motor dysfunction in untreated LXR��/� mice begins at �6months of age, it appears that �-sitosterol accentuates andaccelerates the degeneration that occurs in LXR��/� mice.Elevated levels of plant sterols occur in the brains of ABCG5�/�

or ABCG8�/� mice (17). Although high levels of plant steroldisrupt cholesterol metabolism in extra-cerebral tissues, accu-mulation of �-sitosterol has no detectable effect on overall braincholesterol metabolism (17).

Our study suggests that when LXR� signaling is abnormal,ingestion of �-sitosterol can lead to neuronal damage in bothspinal cord and substantia nigra, thus �-sitosterol could be acommon etiological agent in both PDC and ALS. ALS-PDC isan incurable disease of still unknown etiology. In Guam, PDCand ALS often occur in the same individual and the samefamilies (37).

Histochemical studies with antibodies specific for MAP2B,ubiquitin, and TDP-43 showed that loss of LXR� led to patho-logical changes characteristic of those seen in ALS. Unlike ALS,there was no evidence of activation of immune system in thespinal cord. However, in the substantia nigra there was a clearinvolvement of the immune system with an increase in thenumber of activated microglia.

Recently, Vaya and Schipper (38) have shown that oxysterol-mediated activation of LXR induces ApoE biosynthesis inastrocytes. They suggest that this is a mechanism for removal andredistribution of cholesterol, which would be of benefit inneurodegenerative diseases associated with cholesterol accumu-lation in neurons. Similarly, Zelcer et al. (39) have suggested thatLXRs may be targets in treatment of Alzheimer’s diseasebecause of their ability to modulate both lipid metabolism andinflammatory gene expression.

Our study shows that the use of LXR ligands may lead to braintoxicity, probably caused by cholesterol depletion. If oxysterolsare protective against cholesterol damage to the brain, why does�-sitosterol, a ligand for LXRs, lead to death of motor neuronsand dopaminergic neurons? The answer to this question prob-ably lies in the specific biological functions of the two receptors,LXR� and LXR�. Knockout of LXR� does not lead to motorneuron disease, but LXR��/� mice are very sensitive to ahigh-cholesterol diet. LXR��/� mice tolerate a high-cholesteroldiet without liver accumulation of cholesterol, but they developmotor neuron disease as they age.

Although it has been known for a long time that cholesterolis synthesized in the brain and is extremely important for brainfunction, in recent years more specific functions of cholesterol inthe brain, and toxicity associated with cholesterol, have beendocumented. Many signaling pathways in the brain occur atcholesterol-rich domains known as lipid rafts. These include:signaling of GABA (40) and glutamate (41), synaptic vesicleturnover (42), BDNF protection of motor neurons (43), func-tioning of the calcium channel alpha2delta-2 subunit (44), andcalcium-dependent neurotransmitter release (45).

Fig. 6. Ubiquitin (A and B) and TDP-43 (C and D) immunoreactivity in thecytoplasm of large motor neurons from spinal cord of 8-month-old male mice.(A and C) Group 1. (B and D) Group 3. There were ubiquitin- and TDP-43immunoreactive neuronal cytoplasmic inclusions in large motor neurons inspinal cords of LXR��/�, but not in WT mice. Similar inclusions were seen in thecytoplasm of �-sitosterol-treated LXR��/� mice but because there were veryfew large motor neurons left in the spinal cord of these mice there is notenough data to make conclusions about the occurrence of cytoplasmic inclu-sions in these neurons. (Scale bars: 10 �m.)

Fig. 7. Measurement of cholesterol (A) and 24-hydroxycholesterol (B) in brainsof 8-month-old male mice. The cholesterol content of the brain was determinedwith a cholesterol quantification kit (BioVision). 24-Hydroxycholesterol was de-termined by isotope-dilution mass spectrometry. (A) Data are expressed as �g ofcholesterol/mg of brain tissue. The values are: group 1, 20.9 � 5.2; group 2, 13.8 �2.3; group 3, 27.3 � 9.6; group 4, 13.7 � 3.3; group 1 vs. group 3, *, P � 0.02; group3 vs. group 4, **, P � 0.004; n � 5 – 14 per group; Student’s t test. (B) Data areexpressed as nanograms of 24-hydroxycholesterol/mg of brain tissue. The valuesare: group 1, 34.26 � 15.50; group 2, 30.54 � 14.45; group 3, 32.79 � 17.02; group4, 47.15 � 7.54; group 3 vs. group 4, *, P � 0.04; n � 5 for each group; Student’st test.

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We speculate that in the absence of LXR� activation of LXR�by �-sitosterol leads to efflux of cholesterol from the brain andneuronal toxicity. High levels of 24-hydroxycholesterol indicatedthat the brain was actively involved in elimination of cholesterol.Because �-sitosterol caused reduction in brain cholesterol in WTmice, prolonged use of LXR ligands may lead to neurotoxicitycaused by cholesterol depletion in neurons even when both LXRreceptors are functional.

Materials and MethodsAnimals. LXR��/� mice were generated by gene targeting as described (26). Allmice used in our study have been fully backcrossed for 10 generations toC57BL/6 background. Mice were housed in the Karolinska University HospitalAnimal Facility (Huddinge, Sweden) in a controlled environment on a 12-hlight/dark illumination schedule and fed a standard pellet diet with waterprovided ad libitum.

Administration of �-Sitosterol. Ten WT and 10 LXR��/� male mice (3 months ofage) were divided into four groups: group 1, WT mice given olive oil only;group 2, WT mice given 42 mg/kg �-sitosterol dissolved in olive oil per day;group 3, LXR��/� mice given olive oil only; group 4, LXR��/� mice given 42mg/kg �-sitosterol dissolved in olive oil per day). Olive oil and �-sitosterol weregiven as a daily oral gavage for 3 weeks. The same experimental design wasused with 5-, 8-, and 16-month-old male mice.

Neuropathology. After 3 weeks of treatment with �-sitosterol, mice weredeeply anesthetized with avertin and perfused with PBS followed by 4%paraformaldehyde (in 0.1 M PBS, pH 7.4), and whole brains and vertebralcolumns were collected. After rinsing in PBS, brains were dehydrated in 30%sucrose-PBS and frozen on dry ice. Brains were cryosectioned in coronalorientation at 40 �m. The spinal cords were dissected out, processed forparaffin embedding, cut in coronal orientation on a rotary microtome at 6 �m,and examined after staining with 0.25% thionin (Nissl staining).

For immunohistochemical studies, the tissue sections were incubated in0.5% H2O2 for 30 min to quench endogenous peroxidases, followed by 0.5%Triton X-100 in PBS for 15 min to permeabilize, and then incubated with 1%BSA /0.1% Nonidet P-40 in PBS for 1 h to block unspecific binding. Sectionswere then incubated with mouse anti-MAP2B (1:100; Transduction Laborato-ries), rabbit anti-TH antibody (1:1,000; Novus Biologicals), mouse anti-TDP43antibody (1:200; Proteintech), and rabbit anti-ubiquitin antibody (1:200; Da-kopatts) in 3% BSA/0.1% Nonidet P-40 for 12 h at 4°C. Sections were thenincubated with biotinylated goat anti-rabbit IgG (1:300; Vector Laboratories)or goat anti-mouse IgG (1:300; Vector Laboratories) for 2 h at 37°C, and thenthe avidin-biotin complex was mixed according to the manufacturer’s instruc-tions (Vector Laboratories) for 1 h at room temperature. After every step,sections were washed with PBS for 45 min. Staining was carried out with3,3�-diaminobenzidine (DAKO). For immunofluorescence, sections were incu-bated with cy3-conjugated anti-mouse IgG for 1 h at room temperature andmounted in Vectashield antifading medium (Vector Laboratories). For micro-glial histochemistry, the sections were incubated with 2% H2O2 for 30 min toquench endogenous peroxidases, followed by 1% BSA with 0.1% NonidetP-40 in PBS for 1 h to block unspecific binding. The sections were incubatedwith biotinylated CD11b (1:100; BD Pharmingen) overnight at 4°C. Afterwashing with PBS for 1 h, the sections were incubated with ABC-cy3 antibodyfor 1 h at room temperature. The sections were examined under a Zeissfluorescence microscope with filters suitable for detecting the fluorescenceCy3 or a light microscope.

In the lateroventral horns of L1 segments of the spinal cord, the number ofmotor neurons was evaluated by counting large Nissl-staining motor neurons

of 35–50 �m. Cell counts were made within an area demarcated by a hori-zontal line drawn through the central canal and encompassing the ventralhorn of the gray matter and at least 15–20 slides were counted per spinal cordspecimen (32). Mean counts for each group were compared by Student’s t test.

Rotating Rod Test. Both LXR��/� and WT mice were placed onto a rod of 1.5cm in diameter that rotated at a constant velocity of 0.000387 � g. Theretention times of mice on the rod were recorded, and mice staying for 90 swere taken from the rod and recorded as 90 s. Each mouse was tested fivetimes each day. In each group, five mice were used and means were comparedby Student’s t test.

Wire-Hanging Test. Mice were placed with their forepaws gripping the middleof a 50-cm-long horizontal metallic wire (2 mm in diameter) that was sus-pended between two rods, 30 cm above a foam mattress. The performance ofthe mice was observed for 30 s in four separate trials. The latency to fall wasrecorded, and the ability to grip the wire was scored according to the follow-ing system: 0, fell off; 1, hung onto the wire with two forepaws; 2, in additionto 1, attempted to climb onto the wire; 3, hung onto the wire with twoforepaws and one or both hind paws; 4, hung onto the wire with all four pawswith tail wrapped around the wire; and 5, escaped to one of the supports.Student’s t test was used to compare means of WT and the other groups.

ABCG5, ABCG8, and NPC1L1 mRNA Levels in Intestine. The jejunum (�5 cmcaudal to the pyloric valve) was collected and frozen in liquid nitrogen. RNAextraction and real-time PCR were performed as described (28). The forwardand reverse primer sequences were obtained from published studies (28, 46).

�-Sitosterol and Cholesterol Concentration in Serum. Mice were lightly anes-thetized with avertin, and blood was collected by cardiac puncture fromanimals. The serum was separated by centrifugation and stored at �80°C.Total cholesterol was determined by colorimetric/enzymatic kits (Roche Diag-nostic) in 2 �l of serum. �-Sitosterol was determined in 10 �l of serum fromeach animal. In brief, sera were hydrolyzed for 1 h, at room temperature, in6.6% KOH/ethanol, after addition of d6-�-sitosterol as internal standard.Plant sterols were thereafter extracted in chloroform. The extracts were thenconverted into trimethylsilyl ether derivatives with hexamethyl-silazane andtrichlorosilane. Finally, plants sterols were separated and analyzed by gaschromatography/mass spectrometry (47–49).

Quantification of Cholesterol and 24-Hydroxycholesterol in Brain. Brain choles-terol was extracted by homogenization with chloroform–Triton X-100 (1%Triton X-100 in pure chloroform) in a microhomogenizer. The mixture wasvortexed and centrifuged at 16,000 � g for 10 min at 4°C. The lower organicphase was collected and air-dried at 50°C, and chloroform traces were re-moved with N2 flow. The cholesterol content was then determined with acholesterol quantification kit (BioVision) according to the manufacturer’sinstructions. 24-Hydroxycholesterol in brain samples was determined by iso-tope-dilution mass spectrometry using deuterium-labeled internal standardsas described (26, 50). Fourteen 8-month-old WT and 14 LXR��/� mice brainswere used for the cholesterol measurement assay. Student’s t test was used tocompare cholesterol and 24-hydroxycholesterol concentration between thegroups.

ACKNOWLEDGMENTS. This work was supported by the Consortium for Re-search into Nuclear Receptors in Development and Aging (an integratedproject funded by the Sixth Framework Programme, contract LSHM-CT-2005-018652), Devnertox, a European Union Grant, the European Union Networksof Excellence CASCADE, Konung Gustaf V och Drottning Victorias Stiftelse,and KaroBio AB.

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