Mitochondrial bioenergetic deficit precedesAlzheimer’s pathology in female mouse modelof Alzheimer’s diseaseJia Yao, Ronald W. Irwin, Liqin Zhao, Jon Nilsen1, Ryan T. Hamilton, and Roberta Diaz Brinton2

Department of Pharmacology and Pharmaceutical Sciences and Program in Neuroscience, Pharmaceutical Sciences Center, University of Southern California,Los Angeles, CA 90033

Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved June 29, 2009 (received for review April 2, 2009)

Mitochondrial dysfunction has been proposed to play a pivotal rolein neurodegenerative diseases, including Alzheimer’s disease (AD). Toaddress whether mitochondrial dysfunction precedes the develop-ment of AD pathology, we conducted mitochondrial functional anal-yses in female triple transgenic Alzheimer’s mice (3xTg-AD) andage-matched nontransgenic (nonTg). Mitochondrial dysfunction inthe 3xTg-AD brain was evidenced by decreased mitochondrial respi-ration and decreased pyruvate dehydrogenase (PDH) protein leveland activity as early as 3 months of age. 3xTg-AD mice also exhibitedincreased oxidative stress as manifested by increased hydrogenperoxide production and lipid peroxidation. Mitochondrial amyloidbeta (A�) level in the 3xTg-AD mice was significantly increased at 9months and temporally correlated with increased level of A� bindingto alcohol dehydrogenase (ABAD). Embryonic neurons derived from3xTg-AD mouse hippocampus exhibited significantly decreased mi-tochondrial respiration and increased glycolysis. Results of theseanalyses indicate that compromised mitochondrial function is evidentin embryonic hippocampal neurons, continues unabated in femalesthroughout the reproductive period, and is exacerbated during re-productive senescence. In nontransgenic control mice, oxidativestress was coincident with reproductive senescence and accompaniedby a significant decline in mitochondrial function. Reproductive se-nescence in the 3xTg-AD mouse brain markedly exacerbated mito-chondrial dysfunction. Collectively, the data indicate significant mi-tochondrial dysfunction occurs early in AD pathogenesis in a femaleAD mouse model. Mitochondrial dysfunction provides a plausiblemechanistic rationale for the hypometabolism in brain that precedesAD diagnosis and suggests therapeutic targets for prevention of AD.

ABAD � aging � bioenergetics � brain hypometabolism � mitochondria

The essential role of mitochondria in cellular bioenergetics andsurvival has been well established (1–3). Previous studies have

suggested that mitochondrial dysfunction plays a central role in thepathogenesis of neurodegenerative disorders, including Alzhei-mer’s disease (AD) (1, 4). Alzheimer’s pathology is accompaniedby a decrease in expression and activity of enzymes involved inmitochondrial bioenergetics, which would be expected to lead tocompromised electron transport chain complex activity and re-duced ATP synthesis (5). Further, in AD there is a generalized shiftfrom glycolytic energy production toward use of an alternative fuel,ketone bodies. This is evidenced by a 45% reduction in cerebralglucose utilization in AD patients (6), which is paralleled bydecrease in the expression of glycolytic enzymes coupled to adecrease in the activity of the pyruvate dehydrogenase (PDH)complex (5). Patients with incipient AD exhibit a utilization ratio of2:1 glucose to alternative fuel, whereas comparably aged controlsexhibit a ratio of 29:1, whereas young controls exclusively useglucose as with a ratio of 100:0 ratio (7). In addition to the loweredmitochondrial bioenergetic capacity, impairment of oxidative phos-phorylation is associated with increased free radical production andthe resultant oxidative damage. Overproduction of reactive oxygenspecies and higher oxidative stress is characteristic of brains fromAD (8). Increased oxidative stress, coupled with dysregulation of

calcium homeostasis and resulting apoptosis of vulnerable neuronalpopulations, are proposed to underlie the loss of synaptic activityand associated cognitive decline (9).

In addition to the mitochondrial dysfunction in the clinicallyconfirmed AD cases, multiple analyses have demonstrated thatmitochondrial dysfunction is a plausible contributing factor in thepathogenesis of sporadic AD. The ‘‘cybrid model’’ of AD hasprovided evidence for mitochondrial dysfunction in AD pathogen-esis (10). AD cybrid cells exhibit decreased COX activity, decreasedmitochondrial membrane potential, decreased mitochondrial mo-bility and motility, increased oxidative stress, over activation ofcaspase-3, and increased A� production (11–13). Further, increasedrisk of AD occurs in offspring of women with AD, suggestinghuman maternal mitochondrial inheritance (14, 15). Collectively,these observations suggest a role for mitochondrial dysfunction inthe pathogenesis of sporadic AD.

To determine whether mitochondrial bioenergetic mechanismsare associated with AD pathogenesis, we assessed mitochondrialfunction in the triple transgenic AD mouse model (3xTg-AD)developed by Oddo, LaFerla, and colleagues (16, 17). This trans-genic mouse strain bears mutations in three genes (human APPSWE,TauP301L, and PS1M146V genes) linked to AD and frontotemporaldementia (FTD) and exhibits an age-related neuropathologicalphenotype, including both amyloid beta (A�) deposition and tauhyperphosphorylation (16, 18). To characterize the change inmitochondrial functions in this model, we conducted biochemicaland functional assays on whole brain mitochondria isolated fromboth 3xTg-AD and nontransgenic (nonTg) female mice at differentages. The results presented herein indicate that mitochondrialdysfunction, especially that leading to compromised energy pro-duction, precedes plaque formation, the hallmark histopathology ofAD. Collectively, current clinical findings and our data are sugges-tive of a potential causal role of mitochondrial dysfunction in ADpathogenesis. From a therapeutic perspective, these data support astrategy that targets mitochondrial bioenergetics to prevent or delaythe development of AD.

ResultsAge-Dependent Development of AD-Like Pathology in Female3xTg-AD Mice. The triple-transgenic AD mouse model has beendemonstrated to exhibit an age-related neuropathological pro-gression pattern (16, 18). To determine the temporal correlation

Author contributions: J.Y., J.N., and R.D.B. designed research; J.Y., R.W.I., and R.T.H.performed research; L.Z. contributed new reagents/analytic tools; J.Y., R.W.I., J.N., R.T.H.,and R.D.B. analyzed data; and J.Y., J.N., and R.D.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1Present address: Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320.

2To whom correspondence should be addressed at: Pharmacology and PharmaceuticalSciences, Pharmaceutical Sciences Center, University of Southern California, 1985 ZonalAvenue PSC502, Los Angeles, CA 90089. E-mail: rbrinton@usc.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903563106/DCSupplemental.

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between mitochondrial dysfunction and AD histopathology, wecharacterized the age-dependent development of amyloid pa-thology in the triple-transgenic mouse model. A� accumulationin the hippocampal CA1 region was assessed in both gonadallyintact 3xTg-AD and nonTg female mice at 3, 6, 9, and 12 monthsof age. We observed minimal A�-immunoreactivity (A�-IR) inthe hippocampal CA1 region in the 3xTg-AD mice at 3 months.A�-IR increases in an age-dependent way in 3xTg-AD mice andat 12 months, overt amyloid plaque formation is observed,whereas no A�-IR is observed in nonTg mice at all age groups(Fig. 1).

Decreased Expression and Activity of Key Regulatory Enzymes ofOxidative Phosphorylation in Female 3xTg-AD Mice. Decreased ex-pression and activity of cytochrome c oxidase (COX) and PDHhave been observed in postmortem brain tissue derived fromAlzheimer’s patients (5). To determine if 3xTg-AD mice recapit-ulated these mitochondrial defects and to identify the temporalcorrelation with AD histopathology, hippocampal proteins wereisolated from another set of intact female 3xTg-AD and nonTg miceat 3, 6, 9, and 12 months of age. Expression of PDH (PDH E1�) andCOX (COX subunit IV) was assessed by western blot analysis. PDHE1� expression in 3xTg-AD mice was decreased relative to age-matched nonTg mice (Fig. 2A; P � 0.05, n � 6). Significantlydecreased PDH E1� expression was evident as early as 3 months ofage and was greatest at 12 months of age (Fig. 2A). Likewise, COXIV expression was decreased in 3xTg-AD mice compared withage-matched nonTg mice and was significantly decreased at 9months of age (Fig. 2B).

To confirm that the changes in protein expression were indicativeof changes in enzyme activity, PDH and COX activities wereassessed in mitochondria isolated from whole forebrain of the sameset of 3xTg-AD and nonTg mice used for the hippocampal PDHE1� and COX IV expression. Both PDH and COX activities weredecreased in the aging 3xTg-AD mice as compared with theage-matched nonTg mice (Table 1). Whereas PDH E1� expressionwas significantly decreased as early as 3 months of age in the3xTg-AD mice, significant decline in PDH activity was first evidentat 9 months of age. The preserved PDH enzyme activity relative tothe PDH E1� subunit expression in the 3- and 6-month-old3xTg-AD mice may be indicative of compensatory up-regulation of

PDH activity by posttranslational modification. As with PDHactivity, the decline in COX activity was also significantly decreasedat 9 months of age (Table 1).

Increased Oxidative Stress in Brain Mitochondria of 3xTg-AD Mice.Mitochondrial dysfunction is associated with oxidative stress anddevelopment of AD neuropathology. To determine the oxidativeload of brain mitochondria in the 3xTg-AD mice, we assessed therate of hydrogen peroxide production and magnitude of lipidperoxidation in mitochondria isolated from whole forebrain offemale 3xTg-AD and nonTg mice at 3, 6, 9, and 12 months of age.The Amplex Red hydrogen peroxide assay was used to deter-mine the rates of hydrogen peroxide production of mitochondriain both state 4 (in the absence of ADP) and state 3 (in thepresence of ADP) respiration. There was an age-associatedincrease in the rate of state 4 hydrogen peroxide production inboth the 3xTg-AD and nonTg mice (Fig. 3; P � 0.05, n � 6).More importantly, there was a significant increase in the rate ofstate 4 hydrogen peroxide production in the 3xTg-AD mice ascompared with age-matched nonTg mice, which was evident asearly as 3 months of age and was most pronounced at 12 monthsof age (Fig. 3; P � 0.05, n � 6).

Production of hydrogen peroxide in the nonTg female miceincreased at 9 months of age to a level comparable to, and thus notsignificantly different from, that generated by comparable aged3xTg-AD mice.

Increased hydrogen peroxide production would be expected tolead to a rise in oxidative damage to cellular components. There-fore, we measured lipid peroxidation as an indicator of overalloxidative stress. Both whole forebrain mitochondria and hippocam-pal lysates were used to determine lipid peroxidation, which yieldedcomparable results. Correlated with the increased rate of hydrogenperoxide production, there was significant age-related increase inlipid peroxidation in both the 3xTg-AD and nonTg mice in bothisolated mitochondria (Fig. 4; P � 0.05, n � 6) and hippocampallysates. Further, there was a significant increase in lipid peroxida-tion in the 3xTg-AD mice as compared with age-matched nonTgmice in both isolated mitochondria (Fig. 4; P � 0.05, n � 6) andhippocampal lysates.

Decreased Brain Mitochondrial Respiratory Efficiency in 3xTg-ADMice. Decreased expression and activity of the key mitochondrialregulatory enzymes, PDH and COX, would be expected to result

Fig. 1. Age-related increase in A�-IR in female3xTg-AD mice. Representative images showA�-IR in hippocampus CA1 region from female3xTg-AD mice at 3, 6, 9, and 12 months andfemale nonTg mice at 12 months (Scale bar, 100�m).

Table 1. Decreased PDH and COX activity in female 3xTg-AD mice

Age, months PDH activity (nmol/min/mg protein) Relative COX activity (normalized to 3m nonTg)

nonTg 3xTg-AD nonTg 3xTg-AD3 116.45 � 9.89 126.2 � 5.46 100 � 7.25 105.8 � 6.076 83.73 � 5.37 76.91 � 5.12 94.81 � 7.48 92.22 � 7.339 107.07 � 6.14 86.94 � 4.54* 100.2 � 11.62 74.65 � 9.48*12 79.43 � 5.66 56.59 � 1.37* 69.1 � 2.60 52.7 � 0.63*

Mitochondrial enzyme function in the aging female nonTg and 3xTg-AD mouse brain. Brain mitochondria isolated from both nonTg and 3xTg-AD mice wereassessed for PDH and COX activity. Relative COX Activity was presented as the relative value normalized to that of 3 month nonTg female mice. Mean � SEM(*, P � 0.05 compared with age-matched nonTg group, n � 6).

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in decreased oxidative phosphorylation and impaired mitochon-drial respiratory efficiency. To determine if oxidative phosphor-ylation was altered in the 3xTg-AD mice, mitochondrial respi-

ration was determined in freshly isolated whole forebrainmitochondria from female 3xTg-AD and nonTg mice at 3, 6, 9,and 12 months of age. Respiratory rate of isolated whole brainmitochondria was first determined using glutamate (5 mM) andmalate (5 mM) as respiratory substrates. ADP addition to themitochondrial suspension initiated state 3 respiration. Additionof the adenine nucleotide transporter inhibitor atractylosidereduced the rate of O2 consumption to that of state 40 respira-tion, limited by proton permeability of the inner membrane. InnonTg group, an age-related decline in the respiratory controlratio (RCR; state 3:state 40) was apparent from 3 to 9 monthsand reached statistical significance at 12 months when comparedwith 3 months. Similarly in 3xTg-AD mice, there was also anage-related decline in RCR, which also reached statistical sig-nificance at 12 months when compared with either 3, 6, or 9months (Fig. 5A; P � 0.05, n � 6). More importantly, comparedwith the age-matched nonTg group, 3xTg-AD mice showeddecreased RCR at each age, and this genotype-related impair-ment of mitochondrial respiration deteriorated with age and wasmost pronounced at 12 months of age (Fig. 5A; P � 0.05, n � 6).

The efficiency of mitochondria can also be assessed by deter-mining the rate of free radical leak, or the percent electron flow thatreduces oxygen to ROS instead of reducing O2 to water by COXenzyme. Similar to the change in RCR, in both 3xTg-AD and nonTgmice, the age-related increase in the free radical leak with malate/glutamate plus ADP (state 3) was apparent and reached statisticalsignificance at 12 months when compared with either 3, 6, or 9months (Fig. 5B; P � 0.05, n � 6). More importantly, the freeradical leak was significantly increased in the 3xTg-AD mice ascompared with age-matched nonTg mice at 3, 6, and 12 months ofage (Fig. 5B; P � 0.05, n � 6). As with hydrogen peroxidegeneration, the lack of statistically significant effect between nonTgand 3xTg-AD females at 9 months of age was because of the rise inthe free radical leak of the nonTg to a level comparable to that ofthe 3xTg-AD mice (Fig. 5B).

To determine the cellular contribution to the mitochondrialdeficits of 3xTg-AD mouse brain, basal cellular respiration andglycolysis in primary neuronal cultures from 3xTg-AD and nonTgmice were harvested on embryonic day 14 and cultured for 1 weekin vitro. Oxygen consumption rates (OCR) and extracellular acid-ification rates (ECAR) were determined using the Seahorse XF-24metabolic flux analyzer. In primary neuronal cultures �80% of theOCR measured was because of oxidative phosphorylation (Fig.6A), and �89% of the ECAR measured was because of lactic acidproduction via glycolysis. Neurons derived from the 3xTg-AD miceexhibited significantly lower OCR relative to the OCR of nonTgneurons (Fig. 6A; P � 0.05, n � 3). Correlated with the decreasedoxygen consumption was increased glycolysis, as evidenced by asignificant increase in the ECAR (Fig. 6C). The addition of theATP synthase inhibitor oligomycin (1 �M) resulted in �60%decrease in OCR in neurons from both 3xTg-AD and nonTg mice

Fig. 2. 3xTg-AD female mice have decreased PDH E1� and COX IV protein levelsrelative to age-matched nonTg female mice. Equal amount of hippocampal (forPDH E1�) and mitochondrial (for COXIV) samples from both 3xTg-AD and nonTgmice of different age groups were loaded onto the gel. Expression of (A) PDH E1�

subunit and (B) COX IV subunit were determined by western blot analysis. Barsrepresent mean relative expression � SEM (*, P � 0.05 compared with age-matched nonTg group; n � 6).

Fig. 3. 3xTg-AD female mice exhibit higher oxidative stress than age-matchednonTg female mice. Whole brain mitochondria were isolated from intact femalemice, and rates of hydrogen peroxide production were determined in state 4respiration by Amplex-Red hydrogen peroxide assay. Bars represent mean hy-drogen peroxide production rates � SEM (*, P � 0.05 compared with age-matchednonTggroup;#,P�0.05comparedbetweendifferentagegroupwithinthe same genotype; n � 6).

Fig. 4. 3xTg-AD female mice exhibit higher lipid peroxidation than age-matched nonTg female mice. Whole brain mitochondria were isolated fromintact female mice, and lipid peroxide levels were determined by the leucom-ethylene blue assay. Bars represent mean � SEM (*, P � 0.05 compared withage-matched nonTg group; n � 6).

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(Fig. 6), indicating that the measured oxygen consumption waslargely driven by oxidative phosphorylation-coupled ATP genera-tion. That the decrease in OCR in response to oligomycin wassimilar in both nonTg and 3xTg-AD groups indicates that thedecline in oxygen consumption was not because of a direct impair-ment of ATP synthesis. The decrease in oxygen consumption inresponse to oligomycin was correlated with an increase in ECAR(Fig. 6C), indicating a shift to ATP production through glycolysis

via the Pasteur effect (19). The addition of a mitochondrialuncoupler (FCCP; 1 �M) resulted in a dramatic increase in OCR,as expected, giving an estimation of the maximal respiratorycapacity of the mitochondria. Both the direct measurement of OCRas well as the percent increase over baseline in response to FCCPwere significantly lower in hippocampal neurons derived from the3xTg-AD mice as compared with the nonTg mice (Fig. 6 A and B;P � 0.05, n � 3). These data suggest an impairment of the reserverespiratory capacity in the 3xTg-AD neurons that would potentiatemitochondrial dysfunction in the face of increasing metabolicdemand. Further, the decreased maximal respiratory capacity isconsistent with the impaired COX activity in the 3xTg-AD mice.The addition of the complex I inhibitor rotenone resulted in afurther reduction in OCR values to approximately 15% of baselinein neurons from both 3xTg-AD and nonTg mice (Fig. 5). The �25%difference in OCR values between oligomycin and rotenone expo-sure in both 3xTg-AD and nonTg neurons indicates that bothgroups had equivalent oxygen consumption because of protonleakage. The residual OCR capacity in the presence of rotenonemost likely represents cellular oxygen consumption by nonmito-chondrial pathways.

Increased Mitochondrial A� Levels in 3xTg-AD Mice. Previous studiesindicated that A� interacts with the mitochondrial protein, A�-binding alcohol dehydrogenase (ABAD), and that binding of A�contributes to mitochondrial dysfunction (20, 21). To determine therelationship between mitochondrial A� and the above observedmitochondrial dysfunction, mitochondrial and hippocampal lysatesfrom female 3xTg-AD and nonTg female mice at 3, 6, 9, and 12months of age were analyzed by western blot for ABAD andmitochondrial A� levels. ABAD protein levels decreased with agein nonTg mice, whereas ABAD increased with age in 3xTg-ADmice, which was clearly apparent and significant by 9 and 12 monthsof age in the 3xTg-AD (Fig. 7A; P � 0.05, n � 6). Likewise, at 9months, there was a significantly higher level of A� oligomer levelin the mitochondria of 3xTg-AD mice as compared with age-matched nonTg mice (Fig. 7B; P � 0.05, n � 6).

DiscussionIncreasing evidence implicates mitochondrial dysfunction in mul-tiple neurodegenerative disorders (22). In this report, we demon-strated that the female 3xTg-AD mouse brain recapitulates multipleindicators of mitochondrial dysfunction found in the human ADpatients, including decreased mitochondrial bioenergetics, in-creased oxidative stress, and increased mitochondrial amyloid loadin the 3xTg-AD mouse model (4, 23). Moreover, mitochondrialdysfunction evident in embryonic neurons was sustained through-out postnatal and reproductive ages and was most apparent afterreproductive senescence at 12 months of age. Of particular impor-tance is that the onset of mitochondrial dysfunction in the 3xTg-ADfemale mouse precedes the onset of plaque formation, the classicalhistopathological marker of AD pathology. Although dysfunctionof each of the individual components was not observed at the same

Fig. 5. 3xTg-AD female mice exhibit de-creased mitochondrial respiration relative toage-matched nonTg female mice. Wholebrain mitochondria were isolated from intactfemale mice, and state 3:state 4 respirationwas determined. Oxygen electrode measure-ments of respiration using isolated brain mi-tochondria from 3xTg-AD and nonTg micewere conducted in the presence of 5 mML-malate, 5 mM L-glutamate, 410 �M ADP toinitiate state 3 respiration, and atractylosideto induce state 40 respiration. Bars represent

the mean � SEM of the RCR (state3:state 4 respiration) (*, P � 0.05 as compared with age-matched nonTg group; #, P � 0.05 compared between differentage group within the same genotype; n � 6).

Fig. 6. Hippocampal neurons derived from 3xTg-AD mouse brain exhibitdecreased mitochondrial respiration and increased glycolysis. Primary embryonicneurons derived from both 3xTg-AD and nonTg mice were cultured in Neuro-basal medium plus B27 supplement for 7 days. OCR and ECAR were determinedusing Seahorse XF-24 Metabolic Flux analyzer. (A) OCRs in primary neurons from3xTg-AD mice (black) have lower basal rates of mitochondrial respiration thanprimary neurons derived from nonTg mice (gray). Vertical lines indicate time ofaddition of mitochondrial inhibitors (A) oligomycin (1 �M), (B) FCCP (1 �M), or (C)rotenone (1 �M). The maximal respiratory capacity (FCCP) is significantly lower inneurons from 3xTg-AD mice than those from nonTg mice. (B) Percent change inmitochondrial respiration in response to mitochondrial inhibitors. Bars representthe mean change in OCR from baseline � SEM (*, P � 0.05 as compared withage-matched nonTg group; n � 3). (C) Temporal bioenergetic profiling (ECAR vs.OCR)ofprimaryhippocampalneuronsafterexposure tomitochondrial inhibitors(A) oligomycin, (B) FCCP, and (C) rotenone (*, P � 0.05 as compared with nonTgcultures; n � 3).

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time, mitochondrial dysfunction is more than the sum of its com-ponents. Sustained disturbances in the pathway balances or func-tional efficiency can lead to systems-level defects not observable inadditional individual components until later. Such early imbalancescan lead to accumulated changes in the mitochondria that lateremerge as observable changes in other aspects of the system.Collectively, our findings indicate a critical link between the trans-genes APPswe, PS1M146V, and TauP301L and mitochondrial dysfunc-tion during early neuropathogenesis.

Both the expression and activity of PDH and COX in 3xTg-ADmitochondria were significantly decreased. Reduced mitochondrialefficiency occurred in neurons as evidenced by the shift fromoxidative phosphorylation to lactic acid producing glycolysis inprimary neurons from 3xTg-AD mice. As oxidative phosphoryla-tion is driven by the activity of terminal enzyme COX and PDHserves as the regulatory switch coupling glucose utilization tooxidative phosphorylation, we propose that in 3xTg-AD mice,deficits in the activity of these 2 enzymes reduces the substrate inputand driving force for oxidative phosphorylation, resulting in in-creased ATP demand via other pathways. This unbalanced meta-bolic state coupled with the oxidative stress because of reducedETC efficiency leads to impaired bioenergetics that impairs neu-ronal function and exacerbates neurodegeneration. The occurrenceof these metabolic impairments before plaque formation recapit-ulates findings derived from Alzheimer brain tissue and indicatesthat mitochondrial dysfunction is an important early factor in thedevelopment of AD-like pathology.

The concurrent increase in glycolysis in 3xTg-AD neurons couldbe a compensatory response in which neurons up-regulate glyco-lytic ATP production to compensate for declining mitochondrialrespiration and OXPHOS energy production. In addition, de-creased PDH function could also contribute to the correlateddecrease in mitochondrial respiration and increase in glycolysis.PDH is the key rate-limiting enzyme in mitochondria to convertpyruvate, the end product of glycolysis, into acetyl-CoA, whichsubsequently condenses with oxaloacetate to initiate the TCA cyclefor energy production. Compromised PDH function will lead to theaccumulation of pyruvate and thus should stimulate anaerobicmetabolism to lactic acid and cause an increase in extracellularacidification, as indicated by the increase in ECAR in neuronsderived from 3xTg-AD neurons. Meanwhile, compromised PDHfunction in 3xTg-AD neurons leads to a deficit in acetyl-CoA andconsequently decreased OXPHOS activity as indicated by thedecrease in OCR in 3xTg-AD neurons. These findings suggest apotential antecedent role of mitochondrial bioenergetic deficits inAD pathogenesis and are consistent with previous positron emis-sion tomography (PET) metabolic analyses in persons with in-creased risk of AD, mild cognitive impairment (MCI), or incipientto late AD, in which decreased glucose uptake and utilization wasdemonstrated as among the earliest symptoms of AD occurring farbefore the onset of AD (24–27). These findings are also consistentwith microarray analyses of aging, incipient AD, and AD humansamples and rodent models demonstrating that genes involved inmitochondrial bioenergetics are among those altered early in AD or

MCI patients (22, 28). Consistent with mitochondrial dysfunction,decreased mitochondrial bioenergetics has been demonstrated tocause amyloid production and nerve cell atrophy (29, 30).

In addition to compromised mitochondrial bioenergetics in3xTg-AD mice, elevated oxidative stress was among the earliestindicators of dysfunction in the female 3xTg-AD mouse model. Thisfinding is in agreement with previous studies from both the ADanimal models and the human subjects that showed elevatedoxidative stress plays an important role in AD pathogenesis (31, 32).The data also suggest that multiple aspects of mitochondrialdysfunction are closely related to the pathogenesis of AD. Oxidativedamage to mitochondrial membranes and proteins is well docu-mented to impair mitochondrial OXPHOS efficiency and result inincreased electron leak as observed by increased hydrogen peroxidelevels and higher oxidative stress (9).

A direct link between A�-induced toxicity and mitochondrialdysfunction in AD has been suggested by the interaction betweenmitochondrial A� and a mitochondrial protein, ABAD(HSD17B10, 17�-hydroxysteroid dehydrogenase) (20, 21). As inprevious reports, our analyses show that the increase in mitochon-drial A� correlated with the increase in ABAD level in the3xTg-AD mouse brain. These observations confirmed the neuro-toxic role of mitochondrial deposits of A�. Nevertheless, the rise inABAD and mitochondrial accumulation of A� was subsequent tothe first symptom of mitochondrial dysfunction, which was thedecline in mitochondrial bioenergetic activity. A decline in mito-chondrial respiration and enzymes required for bioenergetics invivo occurred in 3xTg-AD mice as early as 3 months of age.Supporting the hypothesis of mitochondrial dysfunction as anantecedent event to AD pathology, are the in vitro findings derivedfrom embryonic neurons of 3xTg-AD mouse hippocampus. Em-bryonic hippocampal neurons exhibited significant deficits in mi-tochondrial respiratory function. Although we cannot rule out thepossibility that the transgenes themselves could lead to mitochon-drial dysfunction in the 3xTg-AD neurons, it is likely that decreasedmitochondrial bioenergetics coupled with the increase in oxidativestress contributes to the overproduction of A�, which when bindingto ABAD forms an autocatalytic propagation of ROS furtherinducing mitochondrial dysfunction (33).

In summary, mitochondrial dysfunction and deficits in bioener-getics occur early in pathogenesis and precede the development ofobservable plaque formation in female mouse model of AD.Further, age of reproductive senescence markedly exacerbatedmitochondrial and bioenergetic dysfunction which is coincidentwith marked increases in AD pathology. If mitochondrial dysfunc-tion is a causal link to Alzheimer’s, the susceptibility of mitochon-dria to environmental and genetic risks factors should be a criticalfactor in the development of late onset sporadic AD. This postulateis supported by the relationship between brain hypometabolism andincreased risk of AD in offspring with maternal family history ofAD (14, 27). As the mitochondrial genome is maternally inherited,this provides strong evidence of the potential causal role of mito-chondrial dysfunction in AD pathogenesis. From a therapeuticperspective, the findings of this and other studies indicate a ther-

Fig. 7. Female 3xTg-AD mice have increasedABAD and mitochondrial A� protein levelsrelative to age-matched nonTg female mice.Equal amount of hippocampal (for ABAD)and mitochondrial (for A�) samples fromboth 3xTg-AD and nonTg mice of differentage groups were loaded onto the gel. Expres-sion of ABAD and 16 kDa mitochondrial A�

oligomer were determined by western blotanalysis. (A) 3xTg-AD mice have increasedABAD level than age-matched nonTg mice;

(B) increased mitochondrial 16 kDa A� oligomer in the 3xTg-AD mice at 9 months. Bars represent mean relative expression � SEM (*, P � 0.05 comparedwith age-matched nonTg group; n � 6).

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apeutic strategy to prevent AD by sustaining mitochondrial meta-bolic function. Therapeutics, such as estrogen (4, 34, 35), thatsustain and enhance mitochondrial functions by up-regulating keyregulatory enzymes involved in brain metabolism, efficiency ofmitochondrial bioenergetics whereas suppressing oxidative stresscould prevent late onset AD.

Materials and MethodsTransgenic Mice. Colonies of 3xTg-AD and nonTg mouse strain (C57BL/6/129S;The Jackson Laboratory) (16, 17) were bred and maintained at the University ofSouthern California (Los Angeles, CA). Details are provided in the SI Materials andMethods.

Brain Tissue Preparation and Mitochondrial Isolation. Intact female mice of both3xTg-AD and nonTg groups at different age groups (3, 6, 9, and 12 months) werekilled. Brain mitochondria were isolated following a previously established pro-tocol (36). Detailed methods are provided in the SI Materials and Methods.

Immunohistochemistry. For immunohistochemistry study, animals were killed.Brains were perfused with prechilled PBS buffer and immersion fixed in 4%paraformaldehyde. Fixed brains were sent to Neuroscience Associates (NSA,Knoxville, TN) for coronal sectioning at 35 �m, and then processed for immuno-histochemistry using a standard protocol. Detailed methods are provided in theSI Materials and Methods.

Western Blot Analysis. Protein concentrations were determined using the BCAprotein assay kit (Pierce). Western blot analysis was similar to a published proce-dure with minor modifications (36, 37). Detailed methods are provided in the SIMaterials and Methods.

Enzyme Activity Assay. PDH activity was measured by monitoring the conver-sion of NAD� to NADH by following the change in absorption at 340 nm as

previously described (38). Detailed methods are provided in the SI Materialsand Methods.

Hydrogen Peroxide Production. Therateofhydrogenperoxideproductionbyfreshisolated mitochondria was determined by the AmplexRed Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen) following the manufacturer’s instructions.

Lipid Peroxidation. Lipid peroxides in brain mitochondria and hippocampallysates were measured using the leucomethylene blue assay (5). Detailed meth-ods are provided in the SI Materials and Methods.

Respiratory Measurement. Mitochondrial oxygen consumption was measuredpolarographically using a Clark-type electrode following a previously pub-lished procedure (36, 37). Detailed methods are provided in the SI Materialsand Methods.

Free Radical Leak. Free radical leak was determined as previously described (36,39). Detailed methods are provided in the SI Materials and Methods.

Seahorse XF-24 Metabolic Flux Analysis. Primary hippocampal neurons from day14 (E14) embryos of both 3xTg-AD and nonTg mice were cultured, and metabolicflux analysis of neurons derived from both 3xTg-AD and nonTg mice was per-formed according to the manufacturer’s instruction. Detailed methods are pro-vided in the SI Materials and Methods.

Statistics. Statistically significant differences between groups were determinedby an ANOVA followed by a Newman-Keuls posthoc analysis.

ACKNOWLEDGMENTS. This work was supported by Grants from the NationalInstitute on Aging 1 PO1 AG026572 Progesterone in Brain Aging and Alzheimer’sDisease (to R.D.B.) and National Institutes of Mental Health 1RO1 MH67159–01(to R.D.B.). R.W.I. was supported by National Institute on Aging training GrantT32-AG000093–24/25 (C.E. Finch, PI). Critical analysis provided by Dr. EnriqueCadenas is gratefully acknowledged.

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