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Huntington disease is a progressive neurodegenerative disorder withonset generally in midlife. The mutation that causes Huntington dis-ease is an expansion of a CAG repeat in the first exon of the geneencoding the protein huntingtin1. Insoluble huntingtin protein aggre-gates have been seen in vitro, in mammalian cells, in transgenic ani-mals and in brain tissues from patients with Huntington disease2–6.Although the causal relationship between aggregate formation anddisease have been controversial7–9, considerable evidence indicatesthat the formation of insoluble protein aggregates is closely tied to thecellular distortions underlying Huntington disease10–15. Thus, theidentification of molecules or proteins that inhibit the formation ofpolyglutamine aggregates might contribute to the treatment andunderstanding of polyglutamine diseases10.

Several small molecules have neuroprotective effects in a transgenicmouse model of Huntington disease. Minocycline and creatine retardthe progression of pathology and delay mortality16,17. A histonedeacetylase inhibitor improves motor impairment18, and Congo redameliorates the disease by inhibiting oligomerization of huntingtin19.Although the potential therapeutic value of small molecules that pre-vent the formation of polyglutamine aggregates is widelyaccepted20–22, the extreme insolubility of expanded polyglutaminesmakes it difficult to prepare polyglutamine-containing proteins on alarge scale and to search for inhibitors of protein aggregation by invitro high-throughput screening. Recently, we established a large-scalepreparation of a mutant sperm whale myoglobin bearing an expandedpolyglutamine as a molecular model for polyglutamine diseases23,24.The mutant myoglobin reacted with an expanded polyglutamine-spe-

cific antibody in a manner similar to that of the native proteins thatcause polyglutamine diseases, suggestive of a structure for theexpanded polyglutamine quite similar to the structures of the nativeproteins.

Using the mutant myoglobin, we screened for inhibitors of polyg-lutamine-mediated protein aggregation and evaluated candidatemolecules in cellular and transgenic mouse models of Huntingtondisease. In the screening, we focused on small molecules that arenontoxic and can be administered safely and orally. Here, we reportthat various disaccharides reduced the formation of polyglutamineaggregates in vitro and that this correlated with a decrease in celldeath in a cellular model of Huntington disease. Oral administrationof trehalose, the most effective of these disaccharides, inhibited theformation of truncated huntingtin aggregates and improved theassociated motor dysfunction in a transgenic mouse model ofHuntington disease.

RESULTSInhibitory effects of saccharides in vitroTo screen for small molecules that inhibit the formation of polygluta-mine aggregates, we used a mutant myoglobin containing a 35-gluta-mine repeat (Mb-Gln35) that readily formed aggregates uponincubation at 37 °C (ref. 23). We mixed Mb-Gln35 with potentialinhibitors and monitored its aggregation as indicated by absorbance at550 nm. Among a wide variety of compounds tested (more than 200),a disaccharide, trehalose, caused a statistically significant and dose-dependent reduction of aggregation (Fig. 1a). N-acetylgalactosamine

Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako City, Saitama 351-0198, Japan. Correspondence should be addressedto N.N. (nukina@brain.riken.jp).

Published online 18 January 2004; doi:10.1038/nm985

Trehalose alleviates polyglutamine-mediated pathology ina mouse model of Huntington diseaseMotomasa Tanaka, Yoko Machida, Sanyong Niu, Tetsurou Ikeda, Nihar R Jana, Hiroshi Doi, Masaru Kurosawa,Munenori Nekooki & Nobuyuki Nukina

Inhibition of polyglutamine-induced protein aggregation could provide treatment options for polyglutamine diseases such asHuntington disease. Here we showed through in vitro screening studies that various disaccharides can inhibit polyglutamine-mediated protein aggregation. We also found that various disaccharides reduced polyglutamine aggregates and increased survivalin a cellular model of Huntington disease. Oral administration of trehalose, the most effective of these disaccharides, decreasedpolyglutamine aggregates in cerebrum and liver, improved motor dysfunction and extended lifespan in a transgenic mouse modelof Huntington disease. We suggest that these beneficial effects are the result of trehalose binding to expanded polyglutaminesand stabilizing the partially unfolded polyglutamine-containing protein. Lack of toxicity and high solubility, coupled with efficacyupon oral administration, make trehalose promising as a therapeutic drug or lead compound for the treatment of polyglutaminediseases. The saccharide-polyglutamine interaction identified here thus provides a new therapeutic strategy for polyglutaminediseases.

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tetramer (GalNAcGT), an oligosaccharide, also had similar inhibitoryeffects. The effects of trehalose and GalNAcGT were comparable tothat of the QBP1 peptide, which was previously reported to reducepolyglutamine aggregation in vitro25. Because trehalose inhibitedpolyglutamine aggregation, we investigated the ability of other disac-charides to prevent the formation of polyglutamine aggregates. Wefound that disaccharides have a general tendency to decrease thepolyglutamine-mediated aggregation (Fig. 1b).

To investigate the inhibitory mechanism, we examined the stabilityof wild-type myoglobin, of Mb-Gln35 and of Mb-Gln12, which con-tains a shorter 12-glutamine repeat23, in the presence or absence of tre-halose. To evaluate protein stability, we observed the guanidinehydrochloride–induced unfolding of cyanomyoglobins by monitoringthe absorbance at 419 nm23. We calculated the concentration of guani-dine hydrochloride at the midpoint of the unfolding transition (Cm),which is an index of protein stability26 (Table 1). The presence of tre-halose did not affect the Cm value of wild-type myoglobin but resultedin an increase in the Cm of Mb-Gln35 as well as a smaller increase inthe Cm of Mb-Gln12. Thus, trehalose stabilized proteins containing anexpanded polyglutamine.

Effects of various saccharides in mammalian cellsWe next investigated the effects of the small molecules selected by thein vitro screening in stable mouse neuroblastoma Neuro2a cells. Inthese cells, expression of a truncated N-terminal huntingtin (1–90amino acids) containing 60 or 150 glutamines fused to an enhancedgreen fluorescence protein (tNhtt-60Q–EGFP, tNhtt-150Q–EGFP)can be induced by 1 µM ponasterone A, and the cells differentiate inresponse to treatment with 5 mM N6,2’-O-dibutyryladenosine-3′,5′-cyclic monophosphate sodium salt (dbcAMP)27. We found that tre-halose decreased tNhtt-60Q–EGFP aggregates in a dose-dependentfashion without any cellular toxicity (Fig. 1c). GalNAcGT also inhib-

ited the formation of aggregates. We then tested whether other disac-charides could inhibit polyglutamine aggregation. Although the addi-tion of these disaccharides did not alter the expression of tNhtt-EGFPupon treatment with ponasterone A (data not shown), most of the dis-accharides mildly reduced aggregation in tNhtt-60Q–EGFP cells (Fig.1d). To confirm this effect, we transiently overexpressed theEscherichia coli proteins OtsA and OtsB, which produce trehaloseintracellularly28, in tNhtt-150Q–EGFP cells and examined theirinhibitory effects on polyglutamine-induced aggregation. OtsA andOtsB overexpression yielded trehalose in the tNhtt-150Q–EGFP cells(11.8 ± 1.5 nmol/µg protein) and significantly reduced the aggrega-tion (Fig. 1e). This decrease was comparable to that resulting fromtransient overexpression of a molecular chaperone, HDJ-1 (ref. 13;Fig. 1e). We also verified, by western blotting, that the addition of tre-halose and the overexpression of OtsA and OtsB did not induce HDJ-1, HDJ-2 or Hsp70 in the cellular model of Huntington disease (datanot shown).

In addition, we investigated the viability of tNhtt-150Q–EGFP cellsin the presence of inhibitors by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as more tNhtt-150Q–EGFP cells than tNhtt-60Q–EGFP cells die as a result of

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Figure 1 Inhibition of aggregation and cell death by saccharides in vitro. Error bars, s.e.m. n > 3 for each data set. (a,b) Turbidity of Mb-Gln35 solution inthe presence of inhibitors. (c–e) Number of tNhtt-60Q–EGFP cells with green foci (aggregates) in the presence of inhibitors. (f–h) Viability of tNhtt-150Q–EGFP cells in the presence of inhibitors. (a,c,f) (1) 50 µM PBS, (2) 50 µM glucose, (3) 50 µM trehalose, (4) 100 µM trehalose, (5) 1,000 µMtrehalose, (6) 50 µM N-acetylgalactosamine tetramer, (7) 50 µM N-acetylneuraminic acid, (8) 25 µM QBP1. (b,d,g) A series of disaccharides (all at 50µM): (1) PBS, (2) trehalose, (3) sucrose, (4) maltitol, (5) turanose, (6) cellobiose, (7) melibiose, (8) melezitose, (9) mannose. a–d,f,g,*P < 0.05 versusPBS (control). (e) Number of cells with green foci of aggregates in the tNhtt-150Q–EGFP cells in which the indicated protein was overexpressed. *P <0.01 versus LacZ. (h) Viability of tNhtt-150Q–EGFP cells in which the indicated protein was overexpressed. *P < 0.01 versus mock treatment withponasterone A and dbcAMP.

Table 1 Stabilization of partially unfolded Mb-Gln35 by trehalose

Myoglobin Cm (0 µM) Cm (10 µM) Cm (100 µM)

Wild-type 1.66 ± 0.07 1.66 ± 0.04 1.61 ± 0.03

Mb-Gln12 1.04 ± 0.06 1.04 ± 0.03 1.13 ± 0.05

Mb-Gln35 0.85 ± 0.04 1.02 ± 0.03* 1.06 ± 0.03*

Cm values show the midpoints of guanidine hydrochloride–induced unfolding transitionof myoglobins in the absence or presence of trehalose. Values in parentheses indicatethe concentrations of trehalose. Cm values are expressed as mean ± s.e.m. *P < 0.05versus Cm at 0 µM trehalose.

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tNhtt-EGFP expression27. Trehalose produced a dose-dependentincrease in cell viability, as compared with PBS control treatment (Fig.1f). Next, we explored cell survival in the presence of various disaccha-rides. The disaccharides tended to increase cell viability, though theeffects were relatively small (Fig. 1g). To confirm this protective effectof trehalose, we transiently overexpressed OtsA and OtsB in tNhtt-150Q–EGFP cells and found that this enhanced cell viability by morethan 50% (Fig. 1h), similar to the results of transient overexpression ofHDJ-1 (ref. 13).

Beneficial effects of trehalose on transgenic miceTrehalose was the most effective disaccharide among those screened,and its effect was confirmed by overexpressing OtsA and OtsB in thecellular model of Huntington disease (Fig. 1e). Therefore, we testedthe possible neuroprotective effects of trehalose in a mouse model ofHuntington disease, R6/2 transgenic mice29. Trehalose (0.2%, 2% or5%) was added to the drinking water of R6/2 transgenic and wild-typemice, which the mice spontaneously drank. Oral administration oftrehalose reduced the weight loss of transgenic mice (Fig. 2a), whereasit did not affect the body weight of wild-type mice (data not shown).Because this protective effect was most prominent in mice supple-mented with 2% trehalose, we further evaluated the effects of thisdosage of trehalose on the R6/2 transgenic mice. First, we examinedthe influence of trehalose on brain atrophy of the mice. UntreatedR6/2 mice showed dilatation of lateral ventricles resulting from striatalatrophy (ratio of the ventricular area to total cerebrum area, 3.1 ±0.3%), whereas administration of 2% trehalose reduced this ventricu-lar dilatation (ratio 1.8 ± 0.3%, P = 0.008 by unpaired t-test) (Fig. 2b).In addition, the average brain weight was also increased by the oraladministration of trehalose (0.356 ± 0.006 g for untreated R6/2 mice(n = 11), 0.371 ± 0.006 g for R6/2 mice treated with 2% trehalose (n =12)). We next investigated, by immunohistochemistry, the effects oftrehalose on the formation of polyglutamine aggregates in motor cor-tex, striatum and liver29,30. Visualization of intranuclear inclusionswith an antibody to ubiquitin29 showed that ingestion of 2% trehaloseresulted in a substantial decrease in the number of intranuclear aggre-gates (Fig. 2c–e).

We compared the number of polyglutamine aggregates in motor cor-tex, striatum and liver between R6/2 transgenic mice orally adminis-tered 2% trehalose and untreated R6/2 mice at 8 and 12 weeks of age.Trehalose administration unambiguously reduced the number of ubiq-uitin-positive aggregates in the brains of these mice (Fig. 3a,b). We didnot observe any aggregates or histological abnormalities in the brain of8- and 12-week-old wild-type mice supplemented with 2% trehalose(data not shown). For liver, we estimated the number of polyglutamineaggregates in 12-week-old R6/2 transgenic mice, because aggregateswere not clearly detectable in 8-week-old mice30. The number of polyg-

lutamine aggregates in liver was also decreased by the ingestion of 2%trehalose (Fig. 3c). We confirmed the presence of trehalose in thehomogenates of brain (0.11 ± 0.02 nmol/µg protein) and liver (11.6 ±1.1 nmol/µg protein) tissues only in the trehalose-supplemented mice.To gain more insights into the effects of trehalose on aggregation, wecarried out western blotting analysis on R6/2 transgenic mice at age 8weeks as well as at 3 weeks, when mice started to drink the trehalose-containing water. We detected aggregates in the stacking gel foruntreated and 2% trehalose–supplemented 8-week-old R6/2 transgenicmice, whereas we did not observe any oligomeric states of huntingtin31

in the high-molecular-weight region of the separation gel even afteroverexposure of the immunoblot (Fig. 3d,e). The 1C2 antibody raised

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Figure 2 Effects of trehalose on polyglutamine-mediated pathology in vivo.(a) Reduction of body weight loss of R6/2 transgenic mice resulting fromtrehalose treatment. Concentrations of trehalose were 0% (�), 0.2% (�),2% (�) and 5% (�). Error bars, s.e.m. n > 8 for each group of mice. (b)Decrease in striatal atrophy of 12-week-old R6/2 transgenic mice resultingfrom trehalose treatment, as visible in Nissl-stained frozen sections ofcerebrum. Shown are sections from R6/2 transgenic mice treated with 0%(left) or 2% trehalose (right). Scale bar, 400 µm. Values are mean ± s.e.m.(c–e) Inhibition of the formation of huntingtin aggregates by trehalose.Representative images of (c) motor cortex and (d) striatum in cerebrum of 8-week-old R6/2 transgenic mice and of (e) liver of 12-week-old R6/2transgenic mice. Shown are images from R6/2 transgenic mice receiving 0%(left) or 2% trehalose (right). The huntingtin aggregates were visualized withan antibody to ubiquitin. Scale bar, 20 µm.

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against expanded polyglutamine also did not detect any oligomerichuntingtin (data not shown). Oral administration of 2% trehaloseresulted in a reduction in aggregates in the stacking gel (ratio 0.55 ±0.04 for Fig. 3d and 0.57 ± 0.05 for Fig. 3e, by densitometric analysis),which was consistent with the decrease in aggregation seen byimmunohistochemistry (Fig. 3a,b). We observed small amounts ofhuntingtin aggregates in the stacking gel for R6/2 transgenic mice evenat 3 weeks of age (Fig. 3e). To confirm this, we carried out an immuno-histochemical study of 3-week-old R6/2 transgenic mice. We did notdetect ubiquitin-positive aggregates in motor cortex or striatum (datanot shown), as reported previously3. In addition, 3-week-old R6/2transgenic mice did not show any pathogenic symptoms. Next, weassessed neuron viability in the motor cortex and striatum of R6/2transgenic and wild-type mice supplemented with 2% trehalose and incontrols not given trehalose. We calculated the total area of neuronalnuclei in motor cortex and striatum using an antibody that recognizesthe neuron-specific nuclear protein (NeuN)32. The area stained by theantibody to NeuN did not differ statistically either between 8- and 12-week-old R6/2 trehalose-supplemented transgenic mice or between

trehalose-supplemented and unsupplemented transgenic mice (Fig.3f,g). The NeuN-positive area in R6/2 transgenic mice was also similarto that in wild-type mice (Fig. 3f,g), consistent with the observationthat neuron loss was not detected throughout the central nervous sys-tem in R6/2 transgenic mice3,29.

In addition, we assessed the effects of trehalose on the motor func-tion of 7- to 11-week-old R6/2 transgenic mice by rotarod and foot-printing tests. Oral administration of 2% trehalose improved therotarod performance of R6/2 transgenic mice (Fig. 4a) but did notaffect that of wild-type mice (latency time >300 s). This evidence of afavorable effect of trehalose was reinforced by footprinting analysis.Average distance of stride and average width of the walking steps ofR6/2 transgenic mice were increased and decreased by the ingestion oftrehalose, respectively (Fig. 4b,c), showing that trehalose improved thewalking posture of the mice.

Because oral administration of 2% trehalose ameliorated the motordysfunction of R6/2 transgenic mice, we investigated its effect on sur-vival. Oral administration of 2% trehalose extended the lifespan ofR6/2 transgenic mice, and the effect was statistically significant (P =

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e f gFigure 3 Effects of trehalose onaggregation and neuron viability in vivo.Black and gray bars show the data from 8-and 12- week-old R6/2 transgenic mice,respectively. Error bars, s.e.m. *P < 0.01versus 0% R6/2 transgenic mice. (a–c)Number of ubiquitin-positive aggregates(per mm2) in (a) motor cortex, (b) striatumand (c) liver. (d,e) Representative westernblots for the brain homogenates of 3- and8-week-old wild-type (WT) and R6/2transgenic (Tg) mice. Signals weredetected with antibodies to huntingtin or β-tubulin (control) applied to SDS-PAGE gels of (d) total homogenate and (e) its pellet fraction. Molecular sizesare shown at right. (f,g) NeuN-positive area in (f) motor cortex and (g) striatum. n = 9 for mice receiving 0% and n = 8 for mice receiving 2% trehalose.

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Figure 4 Effects of trehalose on motor function and survival in vivo. (a–d) Data are from R6/2 transgenic mice orally administered 0% (�) or 2% trehalose(�). Error bars, s.e.m. Shown are effects of trehalose on (a) rotarod performance, distance of (b) stride and (c) width in walking steps, and (d) lifespan ofR6/2 transgenic mice. a–c, n = 10 for mice receiving 0% and n = 11 for mice receiving 2% trehalose. *P < 0.05 versus mice receiving 0% trehalose. d, n =13 for mice receiving 0% and n = 15 for mice receiving 2% trehalose; P = 0.0015 by log-rank test.

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0.0015 by log-rank test; 107.5 ± 2.3 d for 2% trehalose-supplementedR6/2 transgenic mice, 96.6 ± 2.4 d for unsupplemented R6/2 trans-genic mice) (Fig. 4d).

R6/2 transgenic mice develop diabetes, a feature that mimics the ele-vated diabetes rate in individuals with Huntington disease33,34.We exam-ined the effects of trehalose administration on blood glucose in R6/2transgenic and wild-type mice. We found that the fasting blood glucoselevel of 2% trehalose–supplemented R6/2 transgenic mice (157 ± 7.7 mgdl–1) was not statistically different from that of R6/2 mice not receivingthe supplement (146 ± 6.5 mg dl–1). Wild-type mice ingesting 2% tre-halose also showed a fasting blood glucose level (127 ± 4.5 mg dl–1) com-parable to that of untreated wild-type mice (122 ± 6.3 mg dl–1).

Trehalose orally administered to mice is metabolized to glucose,which might have some effects on R6/2 transgenic mice. Therefore, weassessed the effect of oral administration of 2% glucose on the mice.First, we investigated the frequency of foot clasping, a cardinal pheno-type of R6/2 transgenic mice29, in mice 5 to 7 weeks of age.Administration of 2% trehalose clearly delayed the onset and reducedthe frequency of the foot-clasping posture, whereas administration of2% glucose did not (Fig. 5a,b). Next, we examined the amount ofaggregation in brain and motor function in treated and untreated R6/2transgenic mice. Mice administered 2% glucose showed comparablenumbers of ubiquitin-positive aggregates in motor cortex and striatumand comparable rotarod performance to untreated mice (Fig. 5c–e). Inaddition, the lifespan of mice supplemented with 2% glucose was simi-lar to that of untreated mice (96.1 ± 1.5 d for mice receiving 2% glucose(n = 9), 96.6 ± 2.4 d for unsupplemented transgenic mice (n = 13)).

DISCUSSIONWe have shown here that a variety of disaccharides inhibit polygluta-mine-mediated protein aggregation in vitro and in a cellular model ofHuntington disease. The most effective disaccharide, trehalose, allevi-

ated polyglutamine-induced pathology in vivo. We confirmed thepresence of intracellular trehalose in the cellular and transgenic mousemodels of Huntington disease, and we showed that oral administra-tion of 2% glucose, a metabolite of trehalose, did not ameliorate thepolyglutamine-mediated pathology or extend the lifespan of R6/2transgenic mice. Thus, orally administrated trehalose seems to beresponsible for the neuroprotective effects in vivo. Addition of tre-halose and overexpression of the E. coli trehalose-synthetic proteinsOtsA and OtsB did not change expression levels of heat-shock proteinsin the cellular model of Huntington disease, showing that the trehalosetreatment does not induce cell stress pathways. Instead, the inhibitoryeffects of trehalose on the aggregation of two different polyglutamine-bearing proteins, Mb-Gln35 and truncated huntingtin, indicate thattrehalose may bind directly to expanded polyglutamines.

A complementary inhibition mechanism for trehalose is suggestedby the guanidine hydrochloride denaturation experiment. The pres-ence of trehalose increased the stability of Mb-Gln35 so that it wascomparable to that of Mb-Gln12, which contains a glutamine repeat ofnonpathological length, whereas trehalose did not affect the stabilityof wild-type myoglobin (Table 1). Because trehalose stabilizes proteinsin a partially unfolded state35, the increased stability of partiallyunfolded Mb-Gln35 (ref. 23) caused by trehalose would be expected toreduce the aggregation of Mb-Gln35. Inhibitory effects of trehalose inmammalian cells and in R6/2 transgenic mice might result more fromthe trehalose binding to expanded polyglutamines than from its stabi-lization of truncated huntingtin, because a virtually unstructured,truncated N-terminal huntingtin2 would not refold easily even withaddition of trehalose. Therefore, our cellular studies imply that tre-halose inhibits aggregation by interacting with expanded polygluta-mines. However, because full-length proteins that cause polyglutaminediseases would not be unfolded more than the truncated huntingtin,trehalose could exert beneficial effects on those proteins by stabilizing

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0% trehalose 2% trehalose 2% glucose

Figure 5 Effects of glucose on polyglutamine-mediated pathology in vivo. (a) A typical foot-clasping phenotype of R6/2 transgenic mice with or without2% trehalose or 2% glucose supplementation. 7-week-old R6/2 micesupplemented with 0% (left) and 2% glucose (right) show the foot-claspingposture when suspended by the tail, whereas an R6/2 transgenic mousesupplemented with 2% trehalose (center) holds its hind limbs outward tosteady itself. (b) Frequency of the foot-clasping behavior was investigated for5- to 7-week-old R6/2 transgenic mice. The frequency of clasping was scoredas follows: 3, >10 s; 2, 5–10 s; 1, 0–5 s; 0, 0 s. An average score wascalculated in each group of mice; values are shown as mean ± s.e.m. Filled circle, no supplement; filled triangle, 2% trehalose; open triangle, 2% glucose. n = 7 for 0% mice, n = 9 for 2% trehalose–supplemented mice,n = 14 for 2% glucose–supplemented mice. (c,d) Number of ubiquitin-positive aggregates (per mm2) in (c) motor cortex and (d) striatum of 7-week-old R6/2transgenic mice. n = 5 for mice receiving no supplement, n = 5 for mice receiving 2% trehalose, n = 6 for mice receiving 2% glucose. (e) Effects of glucose onrotarod performance (s) for 7-week-old R6/2 transgenic mice. n = 10 for mice receiving no supplement, n = 11 for mice receiving 2% trehalose, n = 11 formice receiving 2% glucose. *P < 0.05 versus 0% R6/2 transgenic mice.

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their tertiary structures, as it does that of partially unfolded Mb-Gln35. Thus, trehalose could inhibit protein aggregation at the initialstage of aggregate formation by increasing the stability of polygluta-mine-containing proteins.

Oral administration of trehalose reduced the brain atrophy,improved the motor dysfunction and extended the lifespan of R6/2transgenic mice. In a previous study, Congo red delayed the diseaseprocess of R6/2 transgenic mice when administrated by intraperi-toneal or intracerebral infusion14. In our study, we observed compara-ble protective effects on motor function and survival in R6/2transgenic mice that spontaneously ingested trehalose, which is non-toxic, in drinking water. The alleviation of polyglutamine-mediatedpathology by trehalose correlated with a decrease in the number ofintranuclear aggregates. Although there is still much debate as towhether intranuclear inclusion is crucial for pathophysiology7–9, ourdata show that polyglutamine aggregates are important in the patho-genesis of Huntington disease19,36 and that the inhibition of aggrega-tion can lead to amelioration of polyglutamine-mediated pathology10.A previous study showed that translocation of aggregated huntingtinto the nucleus is essential to pathogenesis37. Such translocation ofhuntingtin could also be inhibited by trehalose, because the increase instability of a polyglutamine-containing protein mediated by trehalosewould make the protein more resistant to proteolysis by cas-pases11,16,19,38 and eventually prevent translocation of the cleaved frag-ment into the nucleus.

Western blotting and immunohistochemical analyses showed thatnon-ubiquitinated or non-inclusion aggregates had begun to form at 3weeks, when mice began to drink the trehalose-containing water.Therefore, trehalose may reduce aggregation by preventing furtherassembly of truncated huntingtin, rather than by reversing aggregateformation after it has occurred. A similar decrease in the number ofaggregates in the brain and liver despite different intracellular tre-halose concentrations might be ascribed to a different response systemto trehalose in those tissues, because the brain has a quite different cel-lular organization, such as neuron and glia, from liver. We confirmedthat substantial trehalose was present in the Neuro2a cells overexpress-ing OtsA and OtsB, indicating that intracellular trehalose is probablynot rapidly metabolized to glucose in neurons. Therefore, even in lowconcentrations trehalose could have beneficial effects on the brain,though we cannot completely rule out the possibility that the allevia-tion of pathology may be related to effects of trehalose outside thebrain. A possible adverse effect of trehalose on glucose metabolism wasalso explored in R6/2 transgenic mice. Although this will need to beassessed carefully in clinical tests for Huntington disease patients, theoral administration of trehalose did not affect the fasting blood glu-cose of R6/2 transgenic mice. On the basis of the present study, weexpect that the saccharide-polyglutamine interaction will open a newavenue for the treatment of polyglutamine diseases.

METHODSIn vitro and cell culture experiments. Mb-Gln35, Mb-Gln12 and wild-typemyoglobin were expressed in TB1 E. coli and purified, as previouslydescribed23. 150 µM Mb-Gln35 solutions in 50 mM potassium phosphatebuffer (pH 7.0) were incubated at 37 °C with or without 50 µM of inhibitors.We recorded absorbances of the Mb-Gln35 solutions, as an indication of aggre-gation, at 550 nm at appropriate time points. The guanidinehydrochloride–induced unfolding experiments were carried out with UV-visi-ble spectroscopy, as reported previously23.

In the Neuro2a cellular model of Huntington disease27, in which tNhtt-EGFP expression is induced and cells are differentiated, inhibitors were addedto culture media to a final concentration of 50 µM using a TransPort TransientPermeabilization kit (Life Technologies). After 3 d, we counted aggregate-posi-

tive cells among at least 300 GFP-expressing cells and normalized the numberin each set of inhibitor-treated cells with respect to that in PBS-treated cells.Viability of tNhtt-150Q–EGFP cells was evaluated by MTT assay27. To expressE. coli OtsA and OtsB in Neuro2a cells, an IRES bicistronic expression vector(Clontech) encoding both otsA and otsB was constructed and the vector (10 µg)was transfected into tNhtt-150Q–EGFP cells in 60-mm tissue culture plates13.pcDNA3.1-LacZ, pcDNA3.1– HDJ-1 and empty IRES (mock) vectors (2 µg)were also transfected as controls. We counted aggregate-positive cells after 24 hof treatment with 0.1 µM ponasterone A and 5 mM dbcAMP. Cell viability wasestimated by MTT assay. Concentration of trehalose in the lysate of theNeuro2a cells overexpressing OtsA and OtsB was determined by converting tre-halose to glucose with trehalase (Sigma), which was measured by a glucoseassay kit (Sigma)39. The concentration was normalized by using total proteinconcentration in the cell lysate, which was determined by Bradford assay.Statistical analyses were based on one-factor ANOVA and Bonferroni’s multiplecomparison test using StatView 5.0 (SAS Institute).

R6/2 transgenic and wild-type mice. Heterozygous huntingtin exon-1–trans-genic mice of strain R6/2 (bearing 145 CAG repeats) were obtained from TheJackson Laboratory. Trehalose-containing water was given to R6/2 transgenicand wild-type mice by spontaneous oral administration starting at ∼ 21 d of ageand continuing until the day the mice were killed. R6/2 transgenic and wild-type mice were perfused transcardially with PBS (for determination of tre-halose concentration and western blotting) followed by 4% bufferedparaformaldehyde (for immunohistochemistry). The concentration of tre-halose in brain and liver homogenates was determined by the same method asthat in the Neuro2a cells overexpressing OtsA and OtsB (above). Blood glucosein 6-week-old R6/2 transgenic and wild-type mice (n > 6 for each mice group)was measured after 6 h of fasting with Glutest ace R (Sanwa Kagaku KenkyushoCo.). Mouse experiments were approved by the animal experiment committeeof the RIKEN Brain Science Institute.

Immunohistochemistry and behavioral tests of mice. Serial-cut frozen sec-tions (bregma 0.20–0.50 mm) were used for Nissl staining and immunohisto-chemistry with antibody to ubiquitin (Dako) or NeuN (Chemicon). The areasof lateral ventricles and cerebra of the same Nissl-stained section were meas-ured with a MacSCOPE program (Mitani) (n = 10 each for 0% and 2% tre-halose–supplemented R6/2 mice). We adjusted the contrast and brightness ofthe digital images so that the anti-ubiquitin antibody stained only intranuclearinclusions (not overall nucleus) or the anti-NeuN antibody stained only neu-ronal nuclei, and we calculated the number of ubiquitin-positive aggregatesand the area of NeuN-positive nuclei with MacSCOPE. For the rotarod test,mice were placed on a rotating rod (3.5 rpm), and the rotating speed was lin-early increased to 35 rpm in 300 s (for 7- to 11-week-old mice) and continuedat 35 rpm until 600 s (for 4-week-old mice). Because the latency time of 4-week-old mice was highly dependent on body weight rather than on the pathol-ogy of the mice, we excluded a group of light mice that showed a time of 600 sfrom the statistics. For the footprinting test, steps of the hind paws wererecorded with ink during walking and digital images of the walking steps wereanalyzed. For the clasping test, 5- to 7-week-old mice were suspended by the tailfor 30 s and the frequency of the foot-clasping posture was scored. Statisticalanalyses were done by repeated-measures ANOVA and Bonferroni’s multiplecomparison test and survival curves were analyzed by the Kaplan-Meiermethod and log-rank test, with Statview 5.0.

Western blotting. We prepared brain homogenates of mice (n = 5 for 3-week-old mice, n = 4 for 8-week-old mice) with 50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1% Triton X-100, 1 mM PMSF and Complete protease inhibitor cocktail(Roche), and centrifuged it at 300g for 5 min. The precleared lysate (totalhomogenate) was further spun at 15,000g for 15 min (pellet fraction). Weloaded 50 µg of total protein in each lane of an SDS-PAGE gel (4% stack-ing–10% separation) and used primary antibodies to huntingtin (EM48)(Chemicon), expanded polyglutamine (1C2) (Chemicon) and β-tubulin(Stermberger Monoclonals).

ACKNOWLEDGMENTSWe thank Y. Yamada (RIKEN Brain Science Institute) for maintenance of mice, N.Yoshiki and Y. Obata (RIKEN Tsukuba institute) for instruction on ovarian

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154 VOLUME 10 | NUMBER 2 | FEBRUARY 2004 NATURE MEDICINE

transplantation of R6/2 transgenic mice, and S. Sligar (University of Illinois) forallowing us to use the wild-type myoglobin expression plasmid. This study waspartly supported by grants-in-aid from the Ministry of Education, Culture, Sports,Science and Technology (M.T. and N.N.) and of Health, Labour and Welfare(N.N), Japan, and by RIKEN President’s Special Research Grant (M.T.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 22 August; accepted 22 December 2003Published online at http://www.nature.com/naturemedicine/

1. Zoghbi, H.Y. & Orr, H.T. Glutamine repeats and neurodegeneration. Annu. Rev.Neurosci. 23, 217–247 (2000).

2. Scherzinger, E. et al. Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90, 549–558 (1997).

3. Davies, S.W. et al. Formation of neuronal intranuclear inclusions underlies the neu-rological dysfunction in mice transgenic for the HD mutation. Cell 90, 537–548(1997).

4. Warrick, J.M. et al. Expanded polyglutamine protein forms nuclear inclusions andcauses neural degeneration in Drosophila. Cell 93, 939–949 (1998).

5. Lunkes, A. & Mandel, J.L. A cellular model that recapitulates major pathogenicsteps of Huntington’s disease. Hum. Mol. Genet. 7, 1355–1361 (1998).

6. DiFiglia, M. et al. Aggregation of huntingtin in neuronal intranuclear inclusions anddystrophic neurites in brain. Science 277, 1990–1993 (1997).

7. Michalik, A. & Van Broeckhoven, C. Pathogenesis of polyglutamine disorders:aggregation revisited. Hum. Mol. Genet. 12, R173–R186 (2003).

8. Mccampbell, A. & Fischbeck, K.H. Polyglutamine and CBP: fatal attraction? Nat.Med. 7, 528–530 (2001).

9. Ross, C.A., Poirier, M.A., Wanker, E.E. & Amzel, M. Polyglutamine fibrillogenesis:the pathway unfolds. Proc. Natl. Acad. Sci. USA 100, 1–3 (2003).

10. Bates, G. Huntingtin aggregation and toxicity in Huntington’s disease. Lancet 361,1642–1644 (2003).

11. Ona, V.O. et al. Inhibition of caspase-1 slows disease progression in a mouse modelof Huntington’s disease. Nature 399, 263–267 (1999).

12. Cummings, C.J. et al. Chaperone suppression of aggregation and altered subcellu-lar proteasome localization imply protein misfolding in SCA1. Nat. Genet. 19,148–154 (1998).

13. Jana, N.R., Tanaka, M., Wang, G.H. & Nukina, N. Polyglutamine length–dependentinteraction of Hsp40 and Hsp70 family chaperones with truncated N-terminalhuntingtin: their role in suppression of aggregation and cellular toxicity. Hum. Mol.Genet. 9, 2009–2018 (2000).

14. Shimohata, T. et al. Expanded polyglutamine stretches interact with TAFII130,interfering with CREB-dependent transcription. Nat. Genet. 26, 29–36 (2000).

15. Perez, M.K. et al. Recruitment and the role of nuclear localization in polygluta-mine-mediated aggregation. J. Cell. Biol. 143, 1457–1470 (1998).

16. Chen, M. et al. Minocycline inhibits caspase-1 and caspase-3 expression anddelays mortality in a transgenic mouse model of Huntington disease. Nat. Med. 6,797–801 (2000).

17. Ferrante, R.J. et al. Neuroprotective effects of creatine in a transgenic mousemodel of Huntington’s disease. J. Neurosci. 20, 4389–4397 (2000).

18. Hockly, E. et al. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor,ameliorates motor deficits in a mouse model of Huntington’s disease. Proc. Natl.

Acad. Sci. USA 100, 2041–2046 (2003).19. Sanchez I., Mahlke, C. & Yuan, J. Pivotal role of oligomerization in expanded polyg-

lutamine neurodegenerative disorders. Nature 421, 373–379 (2003).20. Hughes, R.E. & Olson, J.M. Therapeutic opportunities in polyglutamine disease.

Nat. Med. 7, 419–423 (2001).21. Heiser, V. et al. Inhibition of huntingtin fibrillogenesis by specific antibodies and

small molecules: implication for Huntington’s disease therapy. Proc. Natl. Acad.Sci. USA 97, 6739–6744 (2000).

22. Heiser, V. et al. Identification of benzothiazoles as potential polyglutamine aggre-gation inhibitors of Huntington’s disease by using an automated filter retardationassay. Proc. Natl. Acad. Sci. USA 99, 16400–16406 (2002).

23. Tanaka, M., Morishima, I., Akagi, T., Hashikawa, T. & Nukina, N. Intra- and inter-molecular β-pleated sheet formation in glutamine-repeat inserted myoglobin as amodel for polyglutamine diseases. J. Biol. Chem. 276, 45470–45475 (2001).

24. Tanaka, M. et al. The effects of aggregation-inducing motifs on amyloid formationof model proteins related to neurodegenerative diseases. Biochemistry 41,10277–10286 (2002).

25. Nagai, Y. et al. Inhibition of polyglutamine protein aggregation and cell death bynovel peptides identified by phage display screening. J. Biol. Chem. 275,10437–10442 (2000).

26. Luo, Y. & Baldwin, R.L. Trifluoroethanol stabilizes the pH 4 folding intermediate ofsperm whale apomyoglobin. J. Mol. Biol. 279, 49–57 (1998).

27. Wang, G.H. et al. Caspase activation during apoptotic cell death induced byexpanded polyglutamine in N2a cells. Neuroreport 10, 2435–2438 (1999).

28. Guo, N., Puhlev, I., Brown, D.R., Mansbridge, J. & Levine, F. Trehalose expressionconfers desiccation tolerance on human cells. Nat. Biotechnol. 18, 168–171(2000).

29. Mangiarini, L. et al. Exon 1 of the HD gene with an expanded CAG repeat is suffi-cient to cause a progressive neurological phenotype in transgenic mice. Cell 87,493–506 (1996).

30. Sathasivam, K. et al. Formation of polyglutamine inclusions in non-CNS tissue.Hum. Mol. Genet. 8, 813–822 (1999).

31. Poirier, M.A. et al. Huntingtin spheroids and protofibrils as precursors in polygluta-mine fibrilization. J. Biol. Chem. 277, 41032–41037 (2002).

32. Azzouz, M. et al. Increased motoneuron survival and improved neuromuscular func-tion in transgenic ALS mice after intraspinal injection of an adeno-associated virusencoding Bcl-2. Hum. Mol. Genet. 9, 803–811 (2000).

33. Hurlbert, M.S. et al. Mice transgenic for an expanded CAG repeat in theHuntington’s disease gene develop diabetes. Diabetes 48, 649–651 (1999).

34. Farrer, A. Diabetes mellitus in Huntington’s disease. Clin. Genet. 27, 62–67(1985).

35. Singer, M.A. & Lindquist, S. Multiple effects of trehalose on protein folding in vitroand in vivo. Mol. Cell 1, 639–648 (1998).

36. Kazantsev, A. et al. A bivalent Huntingtin binding peptide suppresses polygluta-mine aggregation and pathogenesis in Drosophila. Nat. Genet. 30, 367–376(2002).

37. Yang, W., Dunlap, J.R. Andrews, R.B. & Wetzel, R. Aggregated polyglutamine pep-tides delivered to nuclei are toxic to mammalian cells. Hum. Mol. Genet. 11,2905–2917 (2002).

38. Wellington, C.L. et al. Caspase cleavage of gene products associated with tripletexpansion disorders generates truncated fragments containing the polyglutaminetract. J. Biol. Chem. 273, 9158–9167 (1998).

39. Kandror, O., DeLeon, A. & Goldberg, A.L. Trehalose synthesis is induced uponexposure of Escherichia coli to cold and is essential for viability at low temperature.Proc. Natl. Acad. Sci. USA 99, 9727–9732 (2002).

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