Corrections

EVOLUTION. For the article ‘‘Distinct roles of the homeotic genesUbx and abd-A in beetle embryonic abdominal appendagedevelopment’’ by David L. Lewis, Mark DeCamillis, and RandyL. Bennett, which appeared in number 9, April 25, 2000, of Proc.Natl. Acad. Sci. USA (97, 4504–4509), the authors note thefollowing correction. In Materials and Methods, we failed to citethe source of the Utxm115 mutant. Utxm115 was isolated in thelaboratory of Dr. Robin Denell by Dr. Susan Brown and

colleagues in a screen for recessive lethals within the homeoticcomplex of Tribolium [see Proc. Natl. Acad. Sci. USA (97,4510–4514)]. We are grateful to Rob Denell and Sue Brown forproviding the Utxm115 strain and for communicating unpublishedresults.Correction published online before print: Proc. Natl. Acad. Sci. USA, 10.1073ypnas.160256197. Text and publication date are at www.pnas.orgycgiydoiy10.1073ypnas.160256197

MEDICAL SCIENCES. For the article ‘‘D-b-Hydroxybutyrate protectsneurons in models of Alzheimer’s and Parkinson’s disease’’ byYoshihiro Kashiwaya, Takao Takeshima, Nozomi Mori, KenjiNakashima, Kieran Clarke, and Richard L. Veech, which ap-peared in number 10, May 9, 2000, of Proc. Natl. Acad. Sci. USA

(97, 5440–5444), the authors note the following correction. Figs.1 and 3 were transposed due to a printer’s error. The correctedfigures and their legends are shown below and on the oppositepage.

Fig. 1. Anti-TH stain of day 7 of rat mesencephalic neuronal culture exposed to MPP1 and ketones for 2 days. (A) Control culture of anti-TH-stainedmesencephalic neuronal cultures. (B) Cultures after addition of 5 mM MPP1, (C) after addition of MPP1 and 4 mM ketone bodies, and (D) after addition of 4 mMketone bodies alone. Addition of 5 mM MPP1 to mesencephalic neuronal cultures resulted in a decrease in TH1 cells, a disappearance of neurites, and a shrinkageof cell body volume. Addition of 4 mM Na D-b-hydroxybutyrate to cultures containing 5 mM MPP1 reversed most of the effects of MPP1. The cell number andcell body volume did not differ significantly from control. (Scale bar 5 20 mm.)

8744 u PNAS u July 18, 2000 u vol. 97 u no. 15

Dow

nloa

ded

by g

uest

on

Nov

embe

r 7,

202

0 D

ownl

oade

d by

gue

st o

n N

ovem

ber

7, 2

020

Dow

nloa

ded

by g

uest

on

Nov

embe

r 7,

202

0 D

ownl

oade

d by

gue

st o

n N

ovem

ber

7, 2

020

Dow

nloa

ded

by g

uest

on

Nov

embe

r 7,

202

0 D

ownl

oade

d by

gue

st o

n N

ovem

ber

7, 2

020

Dow

nloa

ded

by g

uest

on

Nov

embe

r 7,

202

0

MEDICAL SCIENCES. For the article ‘‘Oral administration ofa corticotropin-releasing hormone receptor antagonist sig-nificantly attenuates behavioral, neuroendocrine, and auto-nomic responses to stress in primates’’ by Kamal E. Habib,Katherine P. Weld, Kenner C. Rice, Judy Pushkas, MaribethChampoux, Samuel Listwak, Elizabeth L. Webster, Arthur J.Atkinson, Jay Schulkin, Carlo Contoreggi, George P. Chrou-sos, Samuel M. McCann, Stephen J. Suomi, J. Dee Higley, and

Philip W. Gold, which appeared in number 11, May 23, 2000, ofProc. Natl. Acad. Sci. USA (97, 6079–6084), the authors note thefollowing corrections. The sixth line of the next to last paragraphon page 6080 should state 30 sessions rather than 60, andreference 43 should read Korte, S. M., Bouws, G. A. & Bohus,B. (1993) Horm. Behav. 27, 167–183.Correction published online before print: Proc. Natl. Acad. Sci. USA, 10.1073ypnas.160256297. Text and publication date are at www.pnas.orgycgiydoiy10.1073ypnas.160256297

MEDICAL SCIENCES. For the article ‘‘The IgG Fc receptor,FcgRIIB, is a target for deregulation by chromosomal trans-location in malignant lymphoma’’ by Mary B. Callanan, Pa-tricia Le Baccon, Pascal Mossuz, Samuel Duley, ChristianBastard, Rifat Hamoudi, Martin J. Dyer, Gustav Klobeck,Ruth Rimokh, Jean Jacques Sotto, and Dominique Leroux,which appeared in number 1, January 4, 2000, of Proc. Natl.

Acad. Sci. USA (97, 309–314), the authors note the followingcorrection. The first subheading in the Results section shouldread ‘‘Southern Blot Analysis and Cloning of thet(1;22)(q22;q11) Breakpoint Junction.’’

Fig. 3. The effects on cultured rat hippocampal cells of Ab1–42, ketones, or the combination. (A) The 6-day control cultures of 18-day embryonic rat hippocampaltissue; (B) after 14 h exposure to 5 mM Ab1–42, (C) after exposure to both Ab1–42 and 4 mM D-b-hydroxybutyrate, and (D) the effects of ketone bodies alone.Addition of Ab1–42 resulted in a decrease in neuronal number and number of neurites (B versus A). Addition of ketones to cells exposed to Ab1–42 showed nodecrease in neuron or neurite number, indicating that ketones act as neuroprotective agents against the toxicity of Ab1–42 on cultured hippocampal neurons(C versus B).

PNAS u July 18, 2000 u vol. 97 u no. 15 u 8745

CORR

ECTI

ON

Dow

nloa

ded

by g

uest

on

Nov

embe

r 7,

202

0

D-b-Hydroxybutyrate protects neurons in models ofAlzheimer’s and Parkinson’s diseaseYoshihiro Kashiwaya*, Takao Takeshima*, Nozomi Mori*, Kenji Nakashima*, Kieran Clarke†, and Richard L. Veech‡§

*Division of Neurology, Tottori University Faculty of Medicine, Yonago, 683-8503 Tottori, Japan; †Department of Biochemistry, University of Oxford, OxfordOX1-3QU, United Kingdom; and ‡Laboratory of Membrane Biochemistry and BiophysicsyNational Institute of Alcohol Abuse and Alcoholism, 12501Washington Avenue, Rockville, MD 20852

Communicated by Britton Chance, University of Pennsylvania, Philadelphia, PA, March 7, 2000 (received for review November 13, 1999)

The heroin analogue 1-methyl-4-phenylpyridinium, MPP1, both invitro and in vivo, produces death of dopaminergic substantia nigralcells by inhibiting the mitochondrial NADH dehydrogenase mul-tienzyme complex, producing a syndrome indistinguishable fromParkinson’s disease. Similarly, a fragment of amyloid protein,Ab1–42, is lethal to hippocampal cells, producing recent memorydeficits characteristic of Alzheimer’s disease. Here we show thataddition of 4 mM D-b-hydroxybutyrate protected cultured mesen-cephalic neurons from MPP1 toxicity and hippocampal neuronsfrom Ab1–42 toxicity. Our previous work in heart showed thatketone bodies, normal metabolites, can correct defects in mito-chondrial energy generation. The ability of ketone bodies toprotect neurons in culture suggests that defects in mitochondrialenergy generation contribute to the pathophysiology of both braindiseases. These findings further suggest that ketone bodies mayplay a therapeutic role in these most common forms of humanneurodegeneration.

A lzheimer’s disease affects about 5 million people and Par-kinson’s disease about 500,000 people in the United States

(1). The incidence of Alzheimer’s is expected to increase as thepopulation ages as its prevalence rises from 2.5% of those at 65years of age to 47% of those over 85 years of age (2). Alzheimer’sdisease is multifactorial (3), characterized by loss of recentmemory, decrease in brain acetyl choline (4), and death ofhippocampal neurons. These changes result from the accumu-lation of a proteolytic (5) fragment of the b chain of amyloidprecursor protein, Ab1–42, (6) both intracellularly (7) and extra-cellularly in pathologically characteristic amyloid plaques. Re-cently, immunization against Ab1–42 has been reported to pre-vent pathological change in transgenic mice overexpressingamyloid precursor protein (8). There is no general agreement onthe pathophysiological mechanisms of amyloid toxicity. Atpresent, approximately 20% of cases can be related to abnor-malities of Ab1–42 metabolism because of defects located onchromosome 1, 14, 19, or 21 (9), leaving approximately 80% ofcases caused by other factors. Among those factors increasingamyloid accumulation are: brain trauma (10), ischemia (11),insulin resistance (12), or impairment of brain energy metabo-lism (13, 14).

Parkinson’s disease clinically is characterized by muscle rigid-ity, tremor of the distal extremities, and bradykinesia andpathologically is characterized by eosinophilic Lewy-body inclu-sions comprised of the nucleoprotein a-synuclein and ubiquitinand by death of substantial nigral dopaminergic neurons (15).Parkinson’s disease can be caused by genetic abnormalities,environmental toxins, or infections, and it can be treated, at leasttemporarily by L-dopa administration. Experimentally, a syn-drome indistinguishable from Parkinsonism can be induced byadministration of the heroin analogue 1-methyl-4-phenylpyri-dinium, MPP1 (15), which is taken up by the dopamine trans-porter of dopinergic neurons where it inhibits the activity of themitochondrial NADH dehydrogenase multienzyme complex(EC 1.6.5.3) (16, 17).

Brain in normal adults entirely depends on the metabolism ofglucose for its energy needs, being unable to use exogenous fattyor amino acids. The single exception is the ability of brain toderive a major portion of its energy needs from the metabolismof ketone bodies (18), referred to as ketones. In heart, we haveshown that ketones decreased the need for glycolysis (19),bypassed the blockade of pyruvate dehydrogenase (PDH) mul-tienzyme complex resulting from insulin deficiency, increasedthe concentration of metabolites of the first third of the tricar-boxylic acid (TCA) cycle, reduced the mitochondrial [NAD1]y[NADH], oxidized mitochondrial coenzyme Q, thus increasingthe QyQH2 ratio, increased the DG of ATP hydrolysis, andincreased metabolic efficiency (20, 21). Elevation of bloodketones, the brain’s only alternative to glucose as an energysource (18), has been used for 50 years as a treatment forrefractory epilepsy (22). In light of our findings on the effects ofketones in heart, we therefore asked whether they might beneuroprotective against MPP1 toxicity on cultured mesence-phalic neurons and against Ab1–42 toxicity on hippocampalneurons, both models for neurological disease associated withaging.

Materials and MethodsMesencephalic Culture. Primary serum-free culture of 14-dayembryonic mesencephalic cells were prepared from the ven-tral, medial 1.0 mm3 volume block of tissue comprising themesencephalic dopaminergic region as described (23). Thisdissection technique provides cell populations of .95% neu-rons including 20% dopaminergic neurons, tyrosine hydroxy-lase positive (TH1) cells, and ,5% glial cells. Dissected tissueblocks were dispersed by pipetting in DMEMyF12 medium(Gibco) containing 10% FCS and 17.5 mM glucose to whichwas added 0.01% apo-transferrin, 5 mgyml insulin, 30 nMl-thyroxin, 20 nM progesterone, 30 nM sodium selenite, 100unitsyml penicillin, and 100 mgyml streptomycin. Twenty fivemicroliters of the cell suspension containing 5 3 106 cellsymlwas plated on 8-well chamber slides (LabTek, Nunc), coatedwith poly-D-lysine (Sigma). After 4 h incubation at 37°C, in 5%CO2 at 100% humidity, 375 ml of media was added. After 12 hincubation, the medium was aspirated and changed to serum-free medium, which substituted 0.01% BSA (Fraction V,Sigma) for the FCS. At the third day in culture, Na D-b-hydroxybutyrate (Sigma) was added to half the wells to makea final concentration of 4 mM. At the fifth day in culture, 0,1.0, 5, or 10 mM MPP1 (Research Biochemicals-Sigma) wasadded. Survival of neurons was evaluated at the seventh day inculture by the double immunostaining of anti-TH (Boehr-

Abbreviations: MPP1, 1-methyl-4-phenylpyridinium; TCA, tricarboxylic acid; TH, tyrosinehydroxylase; MAP2, microtubular associated protein 2; PDH, pyruvate dehydrogenase.

§To whom reprint requests should be addressed. E-mail: rlveech@csi.com.

The publication costs of this article were defrayed in part by page charge payment. Thisarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.§1734 solely to indicate this fact.

5440–5444 u PNAS u May 9, 2000 u vol. 97 u no. 10

inger) and anti-microtubular associated protein 2 (MAP2)(Boehringer) as described (24).

Hippocampal Cultures. Hippocampal cells were dissected from18-day embryonic rats for microisland cultures (23) and dis-persed by gentle pipetting in neurobasal media (Life Technol-ogies, Grand Island, NY) and centrifuged at 250 g for 10 min.Cells were suspended in neurobasal media containing 1:50 B27,0.5 mM L-glutamine, 25 mM D,L-glutamate, 100 unitsyml peni-cillin, and 100 mgyml streptomycin at a cell density of 2 3 105

cellsyml. A 20-ml aliquot was placed in an eight-chamber LabTek(Nunc-Nalge) culture dish coated previously with poly-D-lysineand placed in an incubator for 4 h, after which 400 ml of mediawas added. On days 2 and 4, half the media was exchanged. On

day 6, half the media was removed and mixed with 200 ml ofDMEMyF12. Na D-b-hydroxybutyrate was added to the mixedmedia and 200 ml replaced in the well so as to create aconcentration within the well of 4 mM. Twelve hours later, halfof the media was replaced with DMEMyF12 with 100 ml of:media only, media containing ketones, media containing 15 mMfresh Ab1–42 (Bachem), or a combination of the latter two. Thefinal concentration of ketones in the media was 4 mM and ofAb1–42 5 mM. The effect of diluting neurobasal media withDMEMyF12 was to raise the media Na1 concentration from78.4 mM to 139.5 mM, within the physiologically normal rangefor extracellular fluid of 136 to 145 mM. At the same time, theinsulin concentration present in neurobasal media was decreasedto 1y3. These changes of inorganic ions toward more physiolog-ical levels in the media increased the rate of neuronal death. Thecells were incubated from 1–36 h. The cells then were fixed with4% paraformaldehyde in PBS for 10 min, permeabilized with 1%acetic acid in 95% ethanol at 24°C for 15 min, washed threetimes with Dulbecco’s PBS, and blocked with BlockAce (Yuki-jirushi, Tokyo). Neurons were stained with anti-MAP2 for 60min. Unbound antibody was removed by washing with PBS for10 min twice. A total of 150 ml of 753 diluted Vector fluoresceinanti-mouse IgG (Vector Laboratories) was added, and the wellswere shaken in darkness for 1 h. The wells were washed twicewith PBS. Ten minutes later the wells were mounted by usingVectashield mounting medium (Vector Laboratories). For stain-ing of glia, antiglial fibrillary acidic protein (Boehringer) wasused in a similar procedure.

Fig. 1. Anti-TH stain of day 7 of rat mesencephalic neuronal culture exposed to MPP1 and ketones for 2 days. (A) Control culture of anti-TH-stainedmesencephalic neuronal cultures. (B) Cultures after addition of 5 mM MPP1, (C) after addition of MPP1 and 4 mM ketone bodies, and (D) after addition of 4 mMketone bodies alone. Addition of 5 mM MPP1 to mesencephlic neuronal cultures resulted in a decrease in TH1 cells, a disappearance of neurites, and a shrinkageof cell body volume. Addition of 4 mM Na D-b-hydroxybutyrate to cultures containing 5 mM MPP1 reversed most of the effects of MPP1. The cell number andcell body volume did not differ significantly from control. (Scale bar 5 20 mm.)

Table 1. The effects of MPP1 and ketone on culturedmesencephalic neuron count

MPP1, mM

TH1 neurons, ymm2 MAP1 neurons, ymm2

Control Ketones Control Ketones

0 65 6 6 64 6 7 485 6 32 476 6 341 30 6 3 46 6 6* 464 6 23 466 6 305 18 6 3 48 6 5* 514 6 25 549 6 29

10 10 6 3 30 6 6* 437 6 29 540 6 27

Values are mean cell countsymm2 6 SEM (n 5 20). p indicates a significantdifference from control at P , 0.05 as judged by Mann–Whitney U test.

Kashiwaya et al. PNAS u May 9, 2000 u vol. 97 u no. 10 u 5441

MED

ICA

LSC

IEN

CES

ResultsEffects of Ketone Bodies on MPP1 Toxicity in Mesencephalic NeuronalCultures. Addition of 1–10 mM MPP1 to cultured mesencephaliccells for 2 days decreased the mean cell count of TH1 cells at allconcentrations tested (Table 1). Addition of 4 mM of NaD-b-hydroxybutyrate, the reduced form of the ketones, signifi-cantly increased the survival of TH1 neurons at all concentra-tions of MPP1 tested (Table 1). Because MPP1 only acts onneurons with a dopamine transporter, there was no effect ofMPP1 or ketones on the number of MAP2-staining neurons inthese mesencephalic cultures. In addition to decreasing the TH1

cell number, exposure to 5 mM MPP1 decreased the outgrowthof neurites, whereas ketones reversed this effect (Fig. 1).

Table 1 shows that ketones act as neuroprotective agentsagainst the toxicity of MPP1 on TH1 dopaminergic neurons inculture but have no effect on the more numerous MAP21

neurons lacking the dopamine uptake system. Antiglial fibril-lary acidic protein staining indicated that glial cells comprisedfewer than 5% of the total cell number in both types of culture.

The Toxic Effects of Ab1–42 on Hippocampal Neurons in Culture WereReversed by D-b-Hydroxybutyrate. In preliminary experiments, wetried doses of Ab1–42 from 2.5, 5.0, 7.5, to 10 mM. A dose of2.5 mM Ab1–42 resulted in no difference in cell count fromcontrol after 8 h incubation; 5 mM Ab1–42 decreased cell countsfrom 172 to 80 whereas control neurons without Ab1–42 de-creased from 170 to 110 over the same period. A dose of 10 mMAb1–42 decreased cell number from 170 to fewer than 10.Accordingly, we picked 5 mM Ab1–42 to study the effects of

Fig. 2. Time course of the effects of 5 mM Ab1–42, 4 mM ketones, or thecombination on the survival of hippocampal neurons in culture. F, The meancontrol cell numberymm2 with error bar indicating the SEM where n 5 12. Allstatistical tests performed were Mann–Whitney U tests, and significance wastaken to be P , 0.05.E, The mean cell number after exposure to Ab1–42;Œafterexposure to 5 mM Ab1–42 1 4 mM D-b-hydroxybutyrate and ‚ after exposureto 4 mM D-b-hydroxybutyrate alone. Exposure to 5 mM Ab1–42 significantlydecreased the cell number compared with controls at 8 and 14 h as indicatedby #. Addition of 4 mM D-b-hydroxybutyrate to cells exposed to 5 mM Ab1–42

increased the cell number compared with exposure of Ab1–42 alone at 8, 14,and 36 h as indicated by *. Addition of ketone bodies alone increased the cellnumber compared with controls as indicated by 1. Our study thereforeconfirms the previous reports of the toxicity of Ab1–42 to cultured hippocampalneurons (28). In addition we show that ketones not only reverse the toxicity ofAb1–42, but act as a growth factor for neurons in culture.

Fig. 3. Theeffectsonculturedrathippocampal cellsofAb1–42, ketones,or thecombination. (A) The6-daycontrol culturesof18-dayembryonic rathippocampal tissue;(B) after 14 h exposure to 5 mM Ab1–42, (C) after exposure to both Ab1–42 and 4 mM D-b-hydroxybutyrate, and (D) the effects of ketone bodies alone. Addition of Ab1–42

resulted in a decrease in neuronal number and number of neurites (B versus A). Addition of ketones to cells exposed to Ab1–42 showed no decrease in neuron or neuritenumber, indicating that ketones act as neuroprotective agents against the toxicity of Ab1–42 on cultured hippocampal neurons (C versus B).

5442 u www.pnas.org Kashiwaya et al.

ketone bodies because this dose of Ab1–42 gave a 50% decrease incell number in 8 h whereas control neurons decreased only 35%.

The exposure of 6-day cultured hippocampal neurons from18-day-old embryos to 5 mM Ab1–42 for 14 h decreased cellnumber (Fig. 2) and neurite number and length (Fig. 3B) incomparison to control (Fig. 3A). Addition of 4 mM D-b-hydroxybutyrate to cells exposed to Ab1–42 doubled the surviv-ing cell number (Fig. 2) and increased cell size and neuriteoutgrowth compared with cells exposed to Ab1–42. This findingshows that ketones may act as neuroprotective agents againstAb1–42 toxicity (Figs. 2 and 3 B versus C). In addition, exposureof cells to ketone bodies for 14 h increased both surviving cellnumber (Fig. 3) and neurite number (Fig. 3D) compared withcontrol cells (Fig. 3A), suggesting that ketone bodies can act asgrowth factors to neurons in culture.

DiscussionThe protection by ketones of hippocampal neurons exposed toAb1–42 or of mesencephalic neurons exposed to MPP1 suggeststhat mitochondrial dysfunction plays a significant role in both of

these common neurological diseases. MPP1 binds to one of twoubiquinone binding sites of the NADH multienzyme complex(17, 25), which is in the same domain as the rotenone andpiericidin A sites. Inhibition of NADH dehydrogenase, in addi-tion to decreasing cell respiration, decreases mitochondrialproton pumping (16) and increases free radical production, thelatter effect being correlated with cell death (26). The majorsource of mitochondrial reactive oxygen species is the semiqui-none form of reduced coenzyme Q (27). Ketone bodies not onlyreduce mitochondrial [NAD1]y[NADH] but increase mitochon-drial [Q]y[QH2] (20, 21). The oxidation of the coenzyme Qcouple should, by decreasing the semiquinone, decrease freeradical production. In the perfused heart, addition of ketonebodies caused a reduction of the free cytosolic NADP couple(R.L.V., unpublished work), which controls the redox state ofglutathione, the major detoxifying agent for H2O2. The oxidationof the coenzyme Q couple should decrease product inhibition ofNADH dehydrogenase while decreasing free radical production,accounting for ketones’ ability to decrease MPP1 toxicity.

How ketones overcome the toxicity of Ab1–42 is not immedi-ately obvious. There are reports however, suggesting that Ab1–42

Fig. 4. The hypothesized effects of ketones on metabolic blocks induced by Ab1–42 and MPP1. Usually brain entirely depends for energy on the mitochondrialmetabolism of pyruvate produced from glucose by the glycolytic pathway. Ab1–42 is reported to stimulate the phosphorylation of the E1a subunit of PDH byglycogen synthase kinase 3b (28). Phosphorylation of PDH blocks the conversion of pyruvate to acetyl CoA, which is required to fuel the TCA cycle, which providesmitochondrial NADH needed to power electron transport. Ketones provide the only alternative source of acetyl CoA for brain during inhibition of the PDHmultienzyme complex. In so doing, ketone bodies not only increase mitochondrial acetyl CoA, citrate, and the first 1y3 of TCA cycle metabolites but also reducethe free mitochondrial NAD couple and oxidize the mitochondrial coenyzme Q couple, causing an increase in the DG of ATP hydrolysis (21). The oxidation ofthe coenzyme Q couple by ketones would tend to decrease the major source of mitochondrial reactive oxygen species, the semiquinone form of coenzyme Q(27), while at the same time relieving product inhibition of NADH dehydrogenase (EC 1.6.5.3), accounting for the ability of ketones to decrease MPP1 toxicity.

Kashiwaya et al. PNAS u May 9, 2000 u vol. 97 u no. 10 u 5443

MED

ICA

LSC

IEN

CES

activates glycogen synthase 3b kinase (28, 29), which phosphor-ylates the E1a subunit of the pyruvate dehydrogenase (PDH)multienzyme complex. Our findings that ketones can ameliorateAb1–42 toxicity are compatible with the ability of ketones tobypass a block at mitochondrial PDH (Fig. 4). Ketones are thephysiological means of overcoming PDH inhibition, resultingfrom a lack of insulin stimulation (20), and ensure the continuingfunction of the TCA cycle and hence the provision of NADH, themajor substrate required for electron transport and ADP phos-phorylation. Mitochondrial function has increasingly been rec-ognized to play a central role in cell death. Opening of themitochondrial voltage-dependent anion channel by the proapo-ptotic protein Bax is an early event in apoptosis (30). Mutationsin mitochondrial DNA are known to result in a wide variety ofrare neurological diseases with pleotropic manifestations (31).Abnormalities in mitochondrial proteins encoded by genomicDNA in Friedreich’s ataxia lead to increased free radical for-mation and a deficiency of mitochondria ATP production (32).However, an increase in the efficiency of mitochondrial energygeneration has not been thought to be important in the mostcommon neurological diseases. Our observation that ketonesoffer neuroprotection in cell culture models of the two mostcommon degenerative neurological diseases has several impor-tant implications.

Despite the genetic and pathophysiological diversity in theetiology of Alzheimer’s and Parkinson’s diseases, our finding

that ketones can protect neurons in culture models of thesediseases is compatible with previous suggestions that the twoconditions have common features (15). Clinically intermediateforms of dementia, specifically Lewy body dementia, sharecommon features, and Parkinsonism is significantly associatedwith dementia and pathologically characterized by Lewy bodiesin the substantia nigra. The Lewy body aggregates of a-synucleinand ubiquitin, while differing in protein composition from theb-amyloid plaque, suggest that both conditions share a commondefect in protein degradative processing that may be related todefective mitochondrial energy generation. This inference iscompatible with the earlier reports of increasing Ab1–42 depo-sition associated with impairment of energy metabolism (13, 14),hypoperfusion (11), or trauma (10).

Another implication of our finding is that elevation of ketonesmay offer neuroprotection in the treatment or prevention of bothAlzheimer’s disease, where therapy is lacking, and Parkinson’sdisease, where therapy with L-dopa is time limited. The high-fatketogenic diet used in childhood epilepsy may not be suitable foruse in adults because of its atherogenic potential; however,alternative dietary sources of ketones produced biotechnologi-cally (33) may overcome this difficulty and provide benefitwithout the undesirable side effects of current ketogenic diets.

This work was supported by grants from the Ministry of Education,Science and Culture of Japan to K.N. and from BTG PLC to K.C.

1. Price, D. L. (1999) Nature (London) 399, A3–A5.2. Evans, D. A., Funkenstein, H. H., Albert, M. S., Scherr, P. A., Cook, N. R.,

Chown, M. J., Hebert, L. E., Hennekens, C. H. & Taylor, J. O. (1989) J. Am.Med. Assoc. 262, 2551–2556.

3. Selkoe, D. J. (1999) Nature (London) 399, A23–A31.4. Pope, A., Hess, H. H. & Lewin, E. (1965) Trans. Am. Neurol. Assoc. 89, 15–16.5. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P.,

Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., et al. (1999) Science 286,735–741.

6. Hardy, J. A. & Higgins, G. A. (1992) Science 256, 184–185.7. Kuo, Y. M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick,

J. B., Murdoch, G. H., Ball, M. J. & Roher, A. E. (1996) J. Biol. Chem. 271,4077–4081.

8. Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu,K., Huang, J., Johnson-Wood, K., Khan, K., et al. (1999) Nature (London) 400,173–177.

9. Selkoe, D. J. (1997) Science 275, 630–631.10. Graham, D. I., Gentleman, S. M., Nicoll, J. A., Royston, M. C., McKenzie, J. E.,

Roberts, G. W. & Griffin, W. S. (1996) Acta Neurochir. (Wien.) Suppl. 66,96–102.

11. Kalaria, R. N., Bhatti, S. U., Lust, W. D. & Perry, G. (1993) Ann. N.Y. Acad.Sci. 695, 190–193.

12. Kuusisto, J., Koivisto, K., Mykkanen, L., Helkala, F. L., Vanhunen, M.,Kervinen, K., Kesaniemi, Y. A., Riekkinen, P. J. & Laakso, M. (1997) Br.Med. J. 315, 1045–1049.

13. Gabuzda, D., Busciglio, J., Chen, L. B., Matsudaira, P. & Yankner, B. A. (1994)J. Biol. Chem. 269, 13623–13628.

14. Lin, L., Georgievska, B., Mattsson, A. & Isacson, O. (1999) Proc. Natl. Acad.Sci. USA 96, 12108–12113.

15. Dunnett, S. B. & Bjorklund, A. (1999) Nature (London) 399, A32–A39.16. Miyoshi, H., Inoue, M., Okamoto, S., Ohshima, M., Sakamoto, K. & Iwamura,

H. (1997) J. Biol. Chem. 272, 16176–16183.17. Sablin, S. O., Krueger, M. J., Yankovskaya, V. L., Tkachenko, S. E., Razdolsky,

A. N., Bachurin, S. O., Ramsay, R. R. & Singer, T. P. (1996) J. Biochem. Toxicol.11, 33–43.

18. Cahill, G. F., Jr. & Aoki, T. T. (1980) in Cerebral Metabolism and NeuralFunction, eds. Passonneau, J. V., Hawkins, R. A., Lust, W. D. & Welsh, F. A.(Williams & Wilkins, Baltimore), pp. 234–242.

19. Kashiwaya, Y., Sato, K., Tsuchiya, N., Thomas, S., Fell, D. A., Veech, R. L. &Passonneau, J. V. (1994) J. Biol. Chem. 269, 25502–25514.

20. Kashiwaya, Y., King, M. T. & Veech, R. L. (1997) Am. J. Cardiol. 80, 50A–64A.21. Sato, K., Kashiwaya, Y., Keon, C. A., Tsuchiya, N., King, M. T., Radda, G. K.,

Chance, B., Clarke, K. & Veech, R. L. (1995) FASEB J. 9, 651–658.22. Freeman, J. M. & Vining, E. P. G. (1992) Epilepsia 33, 1132–1136.23. Takeshima, T., Shimoda, K., Sauve, Y. & Commissiong, J. W. (1994) Neuro-

science 60, 809–823.24. Takeshima, T., Johnston, J. M. & Commissiong, J. W. (1994) J. Neurosci. 14,

4769–4779.25. Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P. &

Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167–3174.26. Barrientos, A. & Moraes, C. T. (1999) J. Biol. Chem. 274, 16188–16197.27. Chance, B., Sies, H. & Boveris, A. (1979) Physiol. Rev. 59, 527–605.28. Hoshi, M., Takashima, A., Noguchi, K., Murayama, M., Sato, M., Kondo, S.,

Saitoh, Y., Ishiguro, K., Hoshino, T. & Imahori, K. (1996) Proc. Natl. Acad. Sci.USA 93, 2719–2723.

29. Hoshi, M., Takashima, A., Murayama, M., Yasutake, K., Yoshida, N., Ishiguro,K., Hoshino, T. & Imahori, K. (1997) J. Biol. Chem. 272, 2038–2041.

30. Shimizu, S., Narita, M. & Tsujimoto, Y. (1999) Nature (London) 399, 483–487.31. Wallace, D. C. (1999) Science 283, 1482–1488.32. Lodi, R., Cooper, J. M., Bradley, J. L., Manners, D., Styles, P., Taylor, D. J. &

Schapira, A. H. (1999) Proc. Natl. Acad. Sci. USA 96, 11492–11495.33. Gerngross, T. U. (1999) Nat. Biotechnol. 17, 541–542.

5444 u www.pnas.org Kashiwaya et al.