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Neurobiology of Aging 35 (2014) 442.e1e442.e8

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Neurobiology of Aging

journal homepage: www.elsevier .com/locate/neuaging

Mitochondrial DNA sequence associations with dementia and amyloid-b in elderlyAfrican Americans

Gregory J. Tranah a,*, Jennifer S. Yokoyama b, Shana M. Katzman c, Michael A. Nalls d, Anne B. Newman e,Tamara B. Harris f, Matteo Cesari g, Todd M. Manini h, Nicholas J. Schork i, Steven R. Cummings a,Yongmei Liu j, Kristine Yaffe k, for the Health, Aging and Body Composition StudyaCalifornia Pacific Medical Center Research InstituteeSan Francisco, San Francisco, CA, USAbMemory and Aging Center, Department of Neurology, University of CaliforniaeSan Francisco, San Francisco, CA, USAcBuck Institute for Research on Aging, Novato, CA, USAd Laboratory of Neurogenetics, Intramural Research Program, National Institute on Aging, Bethesda, MD, USAeDepartment of Epidemiology, University of Pittsburgh, Pittsburgh, PA, USAf Laboratory of Epidemiology, Demography, and Biometry, National Institute on Aging, Bethesda, MD, USAgGérontopôle, INSERM UMR 1027, Centre Hospitalier Universitaire de Toulouse, Toulouse, FrancehUniversity of Florida, Department of Aging and Geriatric Research, Gainesville, FL, USAi Scripps Translational Science Institute, La Jolla, CA, USAjDivision of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, NC, USAkDepartments of Psychiatry, Neurology, and Epidemiology, University of CaliforniaeSan Francisco and the San Francisco VA Medical Center, San Francisco, CA, USA

a r t i c l e i n f o

Article history:Received 12 February 2013Received in revised form 25 May 2013Accepted 26 May 2013Available online 18 October 2013

Keywords:DementiaMitochondriamtDNAAmyloid-bOxidative stress

* Corresponding author at: California Pacific MedicSan Francisco Coordinating Center, UCSF, 185 Berry StrFrancisco, CA 94107-1728, USA. Tel.: þ1 415 600 7410

E-mail address: gtranah@sfcc-cpmc.edu (G.J. Trana

0197-4580/$ e see front matter � 2014 Published byhttp://dx.doi.org/10.1016/j.neurobiolaging.2013.05.023

a b s t r a c t

Mitochondrial dysfunction occurs early in the course of several neurodegenerative diseases, and ispotentially related to increased oxidative damage and amyloid-b (Ab) formation in Alzheimer’s disease.The goals of this study were to assess mtDNA sequence associations with dementia risk, 10-yearcognitive change, and markers of oxidative stress and Ab among 1089 African-Americans in thepopulation-based Health, Aging, and Body Composition Study. Participants were free of dementia atbaseline, and incidence was determined in 187 (18%) cases over 10 to 12 follow-up years. Haplogroup L1participants were at increased risk for developing dementia (odds ratio ¼ 1.88, 95% confidence interval ¼1.23e2.88, p ¼ 0.004), lower plasma Ab42 levels (p ¼ 0.03), and greater 10-year decline on the DigitSymbol Substitution Test (p ¼ 0.04) when compared with common haplogroup L3. The p.V193I, ND2substitution was associated with significantly higher Ab42 levels (p ¼ 0.0012), and this association waspresent in haplogroup L3 (p ¼ 0.018) but not L1 (p ¼ 0.90) participants. All associations were inde-pendent of potential confounders, including APOEε4 status and nuclear genetic ancestry. Identification ofmtDNA sequence variation associated with dementia risk and cognitive decline may contribute to thedevelopment of new treatment targets and diagnostic tests that identify responders to interventionstargeting mitochondria.

� 2014 Published by Elsevier Inc.

1. Introduction

Mitochondrial dysfunction is an especially important charac-teristic of most late-onset neurodegenerative diseases (Mattson,2000) and is a prominent hallmark of Alzheimer’s disease (AD)(Anglade et al., 1997; Blass et al., 2002; Castellani et al., 2002;Chinopoulos et al., 1999; Corral-Debrinski et al., 1994; Coskunet al., 2010; de la Monte et al., 2000; Dragicevic et al.; Eckert et al.,2003; Gibson et al., 2000; Grazina et al., 2006; Hauptmann et al.,

al Center Research Institute,eet, Lobby 5, Suite 5700, San; fax: þ1 415 514 8150.h).

Elsevier Inc.

2009; Hinerfeld et al., 2004; Hirai et al., 2001; Hsiao et al., 1996;Leuner et al., 2012; Manczak et al., 2006; Mucke et al., 2000; Parket al., 1999; Pruijn et al., 1992; Shi et al., 2008; Yao et al., 2009), themost common type of dementia. Considerable evidence suggeststhat the changes in mitochondrial function are causally linked toseveral early abnormalities that accompany AD and precede bothneuronal loss and amyloid-b (Ab) formation (de la Monte et al.,2000; Hauptmann et al., 2009; Leuner et al., 2012; Yao et al.,2009), as well as the clinical manifestation of AD in humans(Gibson and Shi, 2010). These early alterations to mitochondria,which can induce multiple abnormalities, may present moredesirable therapeutic targets than the reversal of the individualpathologies that occur later in the neurodegenerative process.

G.J. Tranah et al. / Neurobiology of Aging 35 (2014) 442.e1e442.e8442.e2

Several lines of evidence show that key enzymes responsible formitochondrial energy metabolism are severely affected in AD (Blasset al., 2002; Eckert et al., 2003; Gibson et al., 1998; Grazina et al.,2006; Kish et al., 1999; Swerdlow et al., 1997). For example, genescoding for respiratory chain subunits are differentially expressed inAD patients (Gibson et al., 1998; Kish et al., 1999). In addition, it isknown that cytoplasmic hybrids (cybrids) carrying mitochondrialDNA (mtDNA) from AD patients exhibit depressed Complex IVactivity when compared with cybrids prepared with mtDNA fromnon-AD controls, suggesting that the deficit is in part encoded bymtDNA abnormalities (Swerdlow et al., 1997). The brain is partic-ularly susceptible to defective mitochondrial function related tomtDNA mutations (Bishop et al., 2010; Gibson and Shi, 2010). Forexample, mtDNA damage may be a major cause of abnormal reac-tive oxygen species (ROS) production in AD or may increaseneuronal susceptibility to oxidative injury during aging.

Human mtDNA is a maternally inherited 16,569ebase pairloopecontaining genes critical to mitochondrial energy production(Wallace, 2010), and bioenergetic defects resulting from acquiredand inheritedmtDNAmutationsmay be critical for both age-relateddementia and associated neuropathological changes observed inAD (Brown andWallace, 1994; Corral-Debrinski et al., 1994; Coskunet al., 2010; De Vivo, 1993; Graeber et al., 1998; Hutchin et al., 1997;Manczak et al., 2004; Tranah et al., 2012b;Wallace, 2001). Sequencevariation within the 13 mtDNA-encoded oxidative phosphorylation(OXPHOS) genes may have an impact on superoxide production atOXPHOS Complexes I and III through respiratory chain impairment(Niemi et al., 2005), apoptosis (Li et al., 2003), and ATP generationefficiency (Tarnopolsky et al., 2004).

Individual mtDNA mutations have been identified in patientswith AD (Brown et al., 1996; Edland et al., 1996; Edland et al., 2002;Egensperger et al., 1997; Grazina et al., 2005; Grazina et al., 2006;Hutchin and Cortopassi, 1995; Janetzky et al., 1996; Kosel et al.,1994; Lakatos et al., 2010; Lin et al., 1992; Petruzzella et al., 1992;Qiu et al., 2001; Shoffner et al., 1993; Tanno et al., 1998; Tranahet al., 2012b; Tysoe et al., 1996; Wragg et al., 1995); yet, many ofthese studies were small, and most of the identified variants havenot been replicated (Edland et al., 2002; Janetzky et al., 1996; Koselet al., 1994; Petruzzella et al., 1992; Tanno et al., 1998; Tysoe et al.,1996; Wragg et al., 1995). To date, the most comprehensive studiesof mtDNA variation in AD (Lakatos et al., 2010), dementia (Tranahet al., 2012b), and cognitive decline (Tranah et al., 2012b) identi-fied haplogroup and individual variant associations with diseasethat were independent of APOEε4 allele status. The majority of thisprevious work, however, has focused onwhite European and NorthAmerican populations, and little is known about risk factors forunderstudied populations. Identifying risk factors for the AfricanAmerican population is of particular importance, as AfricanAmericans have substantially higher AD rates than white NorthAmericans (Evans et al., 2003; Obisesan et al., 2012; Tang et al.,2001). As the prevalence of AD reaches epidemic proportions inthe United States and worldwide in the coming decades (Hebertet al., 2003), understanding the basis of cognitive impairment iscritical to treating and preventing disease. In the present study, weextend our previous mtDNA work in the Health, Aging, and BodyComposition (Health ABC) Study (Tranah et al., 2012b) by assessingdementia risk and cognitive decline in elderly African Americans,and by examining whether mtDNA variation is associated withcirculating Ab levels and markers of oxidative stress (plasmaoxidized LDL and urinary 8-iso-prostaglandin F2a). We have shownthat greater energy expenditure is associated with a reduced inci-dence of cognitive impairment in older adults from the Health ABCStudy (Middleton et al., 2011) and have documented significantdifferences in metabolic rate among African and European mito-chondrial haplogroups from this study (Tranah et al., 2011, 2012a).

Considering the strong link between metabolic rate and cognitiveimpairment (Middleton et al., 2011), these previous studies providethe impetus for understanding the association between mito-chondrial haplogroups and variants and cognition in late life.Uncovering specific mitochondrial variants that have an impact ondementia risk may lead to the development of new interventions orclinical strategies for improving mitochondrial function anddelaying the onset of disease and cognitive decline, as well as ge-netic tests for identifying individuals who are more or less likely torespond to treatments that target the mitochondria.

2. Method

2.1. Study population

Participants were part of the Health ABC Study, a prospectivecohort study of 3075 community-dwelling man and women ofblack andwhite ethnicities living inMemphis, TN, or Pittsburgh, PA,and aged 70 to 79 years at recruitment in 1996 to 1997 (Rooks et al.,2002). To identify potential participants, a random sample of whiteand all black Medicare-eligible elders, within designated zip codeareas, were contacted. To be eligible, participants had to report nodifficulty with activities of daily living, walking a quarter of a mile,or climbing 10 steps without resting. They also had to be free of life-threatening cancer diagnoses and have no plans to move out of thestudy area for at least 3 years. The sample was approximatelybalanced for sex (51% women), and 41% of participants were black.Participants self-designated race/ethnicity from a fixed set of op-tions (Asian/Pacific Islander, black/African American, white/Cauca-sian, Latino/Hispanic, do not know, other). All eligible participantssigned a written informed consent form, approved by the institu-tional review boards at the clinical sites. This study was approvedby the institutional review boards of the clinical sites and thecoordinating center (University of CaliforniaeSan Francisco).

2.2. Genotyping

Genomic DNA was extracted from buffy coat collected using aPUREGENE DNA Purification Kit during the baseline examination.Genotyping was performed by the Center for Inherited DiseaseResearch (CIDR) using the Illumina Human1M-Duo BeadChip sys-tem. This platform includes 138mtDNA SNPs including themajorityof haplogroup-defining variants (Saxena et al., 2006). Sampleswere excluded from the dataset for the reasons of sample failure,genotypic sex mismatch, and first-degree relative of an includedindividual based on genotype data. Only SNPs with call rate �97%and HardyeWeinberg equilibrium p � 10-6 were analyzed. For Af-rican American Health ABC participants, autosomal genotypes wereavailable on 1,007,948 high-quality autosomal SNPS. Genotypingof 138 mtDNA SNPs was successful for 1089 unrelated individualsof African genetic ancestry and yielded 94 polymorphic sites. Themajor African haplogroups were defined in 1029 African Americanparticipants usingPhyloTree (vanOvenandKayser, 2009): L0 (n¼66,6.4%), L1 (n¼ 188,18.3%), L2 (n¼ 360, 35.0%), and L3 (n¼ 415, 40.3%).Sixty participants were identified as belonging to different rareAfrican or Eurasian haplogroups. Nuclear genetic ancestry wasdetermined using a set of 1332 ancestry informative markers thatestimated the proportion of African and European ancestry in theHealthABCAfricanAmericans aspreviouslydescribed(Aldrichet al.).

2.3. Dementia incidence

All participants were free of dementia at baseline. Incident de-mentia was determined by the date of the first available record of adementia diagnosis over 10 to 12 years of follow-up. Cases were

Table 1Baseline characteristics of dementia case individuals and controls among 1089genotyped African American Health ABC participants

Characteristic No dementia Dementia

n (%) 902 (83) 187 (17)Age at baseline, y, mean (SD) 73.3 (2.8) 74.1 (3.0)APOEe4 carrier, n (%) 295 (33) 88 (48)b

Sex, n (%)Male 393 (44) 73 (39)Female 509 (56) 114 (61)

Haplogroup, na (%)L0 56 (84.85) 10 (15.15)L1 139 (73.94) 49 (26.06)L2 300 (83.33) 60 (16.67)L3 351 (84.58) 64 (15.42)

European nuclear genetic ancestry, % (SE) 20 (12) 21 (12)3MS 86.4 (9.4) 82.9 (12.1)DSST 27.8 (14.5) 23.3 (14.2)Ab42 units 32.6 (9.7) 31.9 (9.8)Ab42/40 0.18 (0.06) 0.17 (0.04)oxLDL units 1.41 (0.84) 1.27 (0.57)8-iso-PGF2a units 753 (469) 793 (566)

Key: Ab42, Plasma mean amyloid-beta-42 (pg/mL; n ¼ 433); Ab42/40, plasma meanamyloid-beta-42/40 ratio (n ¼ 423); DSST, Digit Symbol Substitution Test (n ¼1059); oxLDL, plasma oxidized low density lipoprotein (mg/dL; n ¼ 1027); 3MS,Modified Mini-Mental State Examination (n ¼ 1079); 8-iso-PGF2a, urinary 8-iso-prostaglandin, F2-alpha (pg/mg creatinine; n ¼ 584).

a Numbers do not add up to total because of missing information for haplogroups.b APOEe4 frequency significantly differs between dementia cases and controls,

Fisher’s exact test, p ¼ 0.0003.

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identified through hospital records indicating a dementia-relatedhospital event, either as the primary or secondary diagnosisrelated to the hospitalization, or by record of prescribed dementiamedication (i.e. galantamine, rivastigmine, memantine, donepezil,tacrine).

2.4. Cognitive function testing

The Modified Mini-Mental State Examination (3MS) wasadministered to participants at years 1, 3, 5, 7, 9 and 10. The 3MS is abrief, general cognitive battery with components for orientation,concentration, language, praxis, and immediate and delayed recall(episodic memory) (Teng and Chui, 1987). Possible scores rangefrom 0 to 100, with higher scores indicating better cognitive func-tion. The Digit Symbol Substitution Test (DSST) was administered toparticipants at years 1, 5, 7, 9 and 10. The DSST measures responsespeed, sustained attention, visual spatial skills, and set shifting, allof which reflect executive cognitive function (Beres and Baron,1981; Wechsler, 1981) . The test is reported to distinguish milddementia from healthy aging (Tierney et al., 1987). The DSST score iscalculated as the total number of items correctly coded in 90 sec-onds, with a higher score indicating better cognitive function.Participant-specific slopes of 3MS and DSST scores were estimatedby best linear unbiased predictions using mixed-effects modelswith random intercepts and slopes (Fiocco et al.) in STATA10(StataCorp, College Station, TX).

2.5. Biomarkers

Plasma Ab40 and Ab42 levels were measured by the Mayo Clinic(Jacksonville, FL), using Innogenetics (Ghent, Belgium) INNO-BIAassays in samples obtained during the second Health ABC visit(Yaffe et al.). Plasma oxidized LDL (oxLDL) levels were measured insamples obtained during the first Health ABC visi (Cesari et al.,2005; Njajou et al., 2009) by the Atherosclerosis and MetabolismUnit of the Katholieke Universiteit Leuven as previously described(Holvoet et al., 2006) using a monoclonal antibody (4E6)ebasedcompetition ELISA. Urinary 8-iso-prostaglandin F2a (8-iso-PGF2a)levels were measured in samples obtained at the second HealthABC visit (Cesari et al., 2012) by the Laboratory of Clinical Phar-macology of Eicosanoids and Pharmacodynamic located in theCenter of Excellence on Aging at the “Gabriele D’Annnunzio” Uni-versity Foundation (Chieti, Italy), using previously describedradioimmunoassay methods (Ciabattoni et al., 1987; Wang et al.,1995). Summary statistics for biomarkers are presented in Table 1.

2.6. Statistical analyses

We first analyzed dementia risk among the major African hap-logroups and for each of 41 common variants (minor allele fre-quency [MAF] �5%). Unconditional logistic regression was used toobtain odds ratios (ORs) as estimates of relative risks (hereaftercalled risk) and 95% confidence intervals (CIs) for dementiainvolving haplogroups and common variants. Risk of dementia was

Table 2Odds ratios (ORs) and 95% confidence intervals (CIs) for dementia associated with haplo

Haplogroup Cases (n, %) Controls (n, %) OR (95%

L3 64 (15.42) 351 (84.58) Ref.L2 60 (16.67) 300 (83.33) 1.07 (0.L1 49 (26.06) 139 (73.94) 1.88 (1.L0 10 (15.15) 56 (84.85) 1.00 (0.

a Adjusted for age, sex, and clinic site.b Adjusted for age, sex, clinic site, and APOE*e4 status.

examined for haplogroups L0, L1, and L2 as compared to the hap-logroup L3 reference group. Haplogroup L3 was selected as thereference group because it is the most common African haplogroupin our study and this group gave rise to the major Eurasian hap-logroups from which the vast majority of non-Africans aredescended (Salas et al., 2002). The 94 individual mtDNA variantswere examined for associations with dementia using logisticregression, testing risk associated with the rare allele as comparedwith the common allele. Ab (40, 42, and 42/40), oxLDL, and 8-iso-PGF2awere compared among the common African haplogroups andfor individual mtDNA variants using the generalized linear model.All analyses were adjusted for age, sex, and clinic site using SASversion 9.2 (SAS Institute, Cary, NC). Haplogroup analyses wereadjusted for APOEε4 allele carrier status and estimated nuclearEuropean ancestry in secondary models. To avoid false-positiveresults due to population stratification, analyses involving the 137mtDNA variants were also adjusted for 6 eigenvectors of mito-chondrial genetic ancestry derived from principal componentanalysis (PCA) (Biffi et al., 2010; Patterson et al., 2006; Price et al.,2006, 2010; Reich et al., 2008). In our previous mtDNA seq-uencing work the first 6 eigenvectors have accounted for 71% of thevariance in the mtDNA sequence dataset (Tranah et al., 2011).Mitochondrial PCA has been shown to outperform haplogroup-stratified or adjusted association analyses with no loss in powerfor the detection of true associations (Biffi et al., 2010). Several insilico methods were used to examine mtDNA nucleotide conser-vation [PhastCons (Siepel et al., 2005) and PhyloP (Pollard et al.,

group L subgroups

CI)a p Value OR (95% CI)b p Value

Ref.72e1.57) 0.97 1.05 (0.71e1.55) 0.9323e2.88) 0.004 1.78 (1.15e2.76) 0.00948e2.05) 0.74 1.04 (0.50e2.16) 0.83

Table 3Baseline and 10 year rate of change on the DSST and 3MS tests for haplogroup L subgroups

Haplogroup Baseline DSST, mean (SE) (n ¼ 981) DSST slope, mean (SE) (n ¼ 455) Baseline 3MS, mean (SE) (n ¼ 999) 3MS slope, mean (SE) (n ¼ 421)

L3 27.1 (0.71) 0.012 (0.027)a 85.7 (0.50) �0.016 (0.052)L2 27.2 (0.76) 0.030 (0.030)b 86.2 (0.54) 0.011 (0.057)L1 27.1 (1.07) �0.084 (0.039)a,b 85.3 (0.75) �0.002 (0.075)L0 27.3 (1.84) �0.015 (0.060) 84.9 (1.28) 0.052 (0.12)

All values adjusted for age, sex, clinic site, and APOE*e4 status.Pairwise comparisons.

a p ¼ 0.04 for pairwise comparison.b p ¼ 0.02 for pairwise comparison.

G.J. Tranah et al. / Neurobiology of Aging 35 (2014) 442.e1e442.e8442.e4

2010)] for all variants and to predict the potential functional impactof non-synonymous substitutions on amino acid protein sequences[Sorting Tolerant From Intolerant (SIFT) (Kumar et al., 2009; Ng andHenikoff, 2006), MutPred (Li et al., 2009), and PolyPhen2 (Adzhubeiet al., 2010)].

Table 4Comparison of amyloid-b levels and markers of oxidative damage among AfricanAmerican haplogroups

Haplogroup Ab42(n ¼ 433)

Ab42/40(n ¼ 423)

oxLDL(n ¼ 1,027)

8-iso-PGF2a(n ¼ 584)

L3 33.70 (0.76)a 0.18 (0.005) 1.39 (0.04) 792 (32)L2 32.14 (0.76) 0.18 (0.005) 1.38 (0.04) 742 (36)L1 31.12 (1.09)a 0.17 (0.007) 1.45 (0.06) 757 (47)L0 31.69 (1.77) 0.18 (0.011) 1.23 (0.10) 661 (71)

All values adjusted for age, sex, and clinic site.Key (for pairwise comparisons): Ab42/40, plasma mean amyloid-beta-42/40 ratio;oxLDL, plasma oxidized low density lipoprotein (mg/dL); 8-iso-PGF2a, urinary 8-iso-prostaglandin, F2-a (pg/mg creatinine).

a p ¼ 0.03.

3. Results

Among 1089 genotyped African American Health ABC partici-pants,187 (17%) developed dementia (Table 1). In general, dementiacases were more likely to be APOEε4 allele carriers, but there wereno differences in age, sex, or levels of European nuclear geneticancestry (Table 1). Haplogroup frequencies are consistent withmtDNA sequencing performed by us (Lam et al., 2012) and others(Saxena et al., 2006), and mean European nuclear genetic ancestrydid not differ (p¼ 0.38) among the 4 major African haplogroups: L0(19%), L1 (21%), L2 (20%), L3 (21%). Among the 94 polymorphicmtDNA sites detected after genotyping 138 SNPs, 53 occurred at aMAF of <5% and 41 at MAF of �5%.

Risk of developing dementia among the 4 African sub-haplogroups is reported in Table 2. Carriers of haplogroup L1 hadan increased risk of developing dementia compared with the mostcommon African haplogroup L3 in a model adjusted for age, sex,and clinic site (OR ¼ 1.88, 95% CI ¼ 1.23e2.88, p ¼ 0.004) that wasstatistically significant after adjustment for multiple comparisons(3 haplogroups, critical a ¼ 0.016). Adjustment for either APOEε4allele carrier status (OR ¼ 1.78, 95% CI ¼ 1.15e2.76, p ¼ 0.009) orEuropean ancestry (OR ¼ 1.76, 95% CI ¼ 1.14e2.73, p ¼ 0.010)slightly attenuated the associations, but the results remained sta-tistically significant at an adjusted threshold. Haplogroup L1 par-ticipants experienced a slightly greater 10-year decline in DSST(b ¼ �0.08, � standard error [SE] ¼ 0.04) when compared withhaplogroups L2 (p ¼ 0.02) and L3 (p ¼ 0.04) (Table 3). Adjustmentfor European ancestry and education level did not affect results(data not shown). There were no haplogroup differences in eitherbaseline 3MS or 3MS slopes.

We examined haplogroup differences among the subsets ofparticipants with plasma oxLDL, urinary 8-iso-PGF2a, and plasmaAb40 and Ab42. Among 433 African American participants withplasma Ab42 levels measured, haplogroup L1 participants hadnominally lower (p ¼ 0.03) Ab42 levels when compared with par-ticipants from haplogroup L3 (Table 4). No haplogroup differenceswere identified for oxLDL, 8-iso-PGF2a, and Ab42/40 ratios.

Among the 41 common mtDNA variants genotyped, them.5046G>A variant encoding a p.V193I, ND2 substitution wasassociated with significantly higher (p ¼ 0.001) Ab42 levels afteradjustment for multiple comparisons (critical a ¼ 0.0012). Hap-logroup L carriers of the m.5046A allele (n ¼ 119) had an elevatedmean (SE) Ab42 of 43.0 (3.5) pg/mL when compared with a mean(SE) Ab42 of 31.2 (0.59) pg/mL for carriers of the m.5046G allele(n¼ 968). The m.5046G>Avariant was present only in haplogroupsL1 and L3. We identified a statistically significant (p ¼ 0.047)interaction between the m.5046A variant and haplogroup L1/L3

status in a model that included age, sex, m.5046G>A genotype, L1/L3 status, and an interaction term for m.5046G>A genotype and L1/L3 status. The m.5046A allele was significantly associated withelevated Ab42 levels among haplogroup L3 (p ¼ 0.018) but not L1(p¼ 0.90) participants (Table 5). Specifically, haplogroup L3 carriersof the m.5046A allele had an elevated mean (SE) Ab42 of 43.2 (4.5)pg/mL when compared with a mean (SE) Ab42 of 33.4 (0.80) pg/mLfor carriers of the m.5046G allele. There was no in silico evidencefor nucleotide conservation (phastCons, phyloP) or predictedfunctional significance (SIFT, PolyPhen2, and MutPred) for thep.V193I, ND2 substitution. None of the individual mtDNA variantswere associated with dementia risk, 3MS, or DSST slopes afteradjustment for multiple comparisons.

4. Discussion

Several mechanisms underlie the changes observed in the agingbrain including mitochondrial function and oxidative stress, auto-phagy, and protein turnover (Bishop et al., 2010). Mitochondrialdysfunction, in particular, occurs early in the neurodegenerativeprocess and precedes neuronal loss and early Ab formation in AD(de la Monte et al., 2000; Hauptmann et al., 2009; Leuner et al.,2012; Yao et al., 2009). Several key enzymes responsible for mito-chondrial energy metabolism are severely affected in AD (Blasset al., 2002; Castellani et al., 2002; Eckert et al., 2003; Grazinaet al., 2006; Swerdlow et al., 1997), and the results presentedherein suggest that Complex I genetic variationmay be of particularimportance to the neurodegenerative process. Complex I is a large,multi-subunit, membrane-bound protein that serves as the majorentry point for most electrons into the electron transport chain.This complex is a major contributor to cellular ROS production(Hirst). Inhibition of Complex I leads to increased generation of ROS,decreased ATP levels, and induction of apoptosis (Langston andBallard, 1983; Li et al., 2003; Ramsay and Singer, 1986), all ofwhich play a major role in neurodegeneration. In previous studieslinking Complex I mtDNA sequence data to cognitive function anddisease, non-synonymous ND4 and ND5 substitutions were asso-ciated with AD risk (Lakatos et al., 2010), and we reported ND6associations with decline in 3MS (Tranah et al., 2012b).

Table 5Association among m.5046G>A, p.V193I, ND2, and Ab42 levels among haplogroup L1 and L3 participants

n (%) Haplogroup L1 Ab42 p Value n (%) Haplogroup L3 Ab42 p Value

Mean (SE) Mean (SE)

A 39 (49%) 31.0 (1.66) 5 (3%) 43.4 (4.50)G 40 (51%) 31.3 (1.64) 0.90 156 (97%) 33.4 (0.80) 0.018

All values adjusted for age and sex.

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In the current study, Haplogroup L1 participants were at asignificantly increased risk for developing dementia, experienced asignificant 10-year decline in DSST, and had lower plasma Ab42levels [which has previously been associated with increased risk ofdeveloping AD (Graff-Radford et al., 2007; Lewczuk et al., 2010;Pesaresi et al., 2006) and greater cognitive decline on the 3MS(Yaffe et al., 2011)]. The large number of variants that are closelyassociated with one another and that define haplogroup L1 (char-acterized by Complex I [ND1, ND5, ND6] and Complex IV [COI] vari-ants) (van Oven and Kayser, 2009) complicate interpretation ofhaplogroup association data. In addition, 3 of these haplogroup-defining variants encode amino acid substitutions with possiblefunctional potential: p.Y485H, ND5, p.I166V, ND6, and p.Y496H, COI.We previously observed that European haplogroup T participantswere at a significantly increased risk for developing dementia andthat haplogroup J participants experienced a significant longitudinaldecline in 3MS (Tranah et al., 2012b). Haplogroup T is defined bymultiple variants in the 12S and 16S rRNAs, tRNA Arg, tRNA Thr, ND2,ND5, CytB, andATP6 (vanOven andKayser, 2009)withonly 1 of thesevariants having apparent functional potential: m.4917A>G, whichencodes amino acid substitution p.N150D, ND2 in Complex I. Hap-logroup J is defined by variants in ND5 and the hypervariable region(vanOvenandKayser, 2009). Suchhaplogroupassociations canmaskthe effects of individual nucleotide changes because most mito-chondrial haplogroups can be defined by several control region and/or coding region variants. This implies that functional studies shouldaccount for all mtDNA variants within the mitochondrial genome,especially for haplogroups that can be defined by several variants.

The analysis of mtDNA is further complicated by recurrentmutation at the same mtDNA site across divergent haplogroups,which can sometimes hide diagnostic specificity of a particularvariant. That the samemutations have been observed repeatedly ondifferent mtDNA backgrounds (e.g., haplogroups) has been cited asevidence of convergent adaptive evolution of particular mtDNAmutations (Wallace, 2010). We observed that the m.5046G>Avariant encoding the p.V193I, ND2 substitution was significantlyassociated with Ab42 levels and that this associationwas specific tohaplogroup L3 carriers of the variant but not haplogroup L1 carriers.Indeed, significant differences in the frequency of non-synonymousmutations among haplogroups (Moilanen et al., 2003) suggests thatsome mutations may be non-neutral within specific phylogeneticlineages but neutral within others.

This study has a number of strengths: a well-characterizedpopulation-based African-American cohort with longitudinalassessment of 3MS and DSST; a large sample size for assessingdementia risk and Ab among African mtDNA haplogroups andvariants; measurement of circulating Ab levels, and markers ofoxidative stress. Limited power to detect individual effects of rarevariants is acknowledged. These preliminary results are based on asingle cohort and further studies are needed to confirm thesefindings. In addition, plasma- and urine-derived biomarkers maynot accurately reflect the state of brain levels.

In summary, dementia is a complex neurodegenerative disorderwith a multifaceted genetic and clinical pathogenesis involvingneurodegenerative, vascular, and metabolic causes. Mitochondrialdysfunction underlies the symptoms of many human neurological

disorders, including AD (Mattson, 2000), which suggests the exis-tence of a shared neurodegenerative mechanism operating acrossmultiple pathologies. Identifying dementia-associated OXPHOSComplex I variants may lead to targeted interventions that affectsuperoxide production (Brand, 2010; Li et al., 2003). For example,several natural compounds (Baur and Sinclair, 2006; Baur et al.,2006; Chowanadisai et al., 2010; Davis et al., 2009; Liu et al., 2007;Nogueira et al., 2011; Pratico, 2008; Rasbach and Schnellmann,2008; Rodriguez et al., 2007; Stites et al., 2006; Tauskela, 2007;Timmers et al., 2011), behavioral interventions (Civitarese et al.,2007; Guarente, 2008; Johnston et al., 2008; Lopez-Lluch et al.,2006; Menshikova et al., 2006; Nisoli et al., 2005), and pharmaco-logic agents (Bar-Am et al., 2009; Weinreb et al., 2008a; Weinrebet al., 2008b; Youdim and Buccafusco, 2005) target the mitochon-dria and have been shown to induce mitochondrial biogenesis andincrease electron transport chain activity. Further studies confirm-ing our findings may provide detailed clues about the mechanismsof disease related to specific mitochondrial variants, and ultimatelymay contribute to the development of genotype-specific thera-peutic interventions for delaying the onset of disease and cognitivedecline.

Disclosure statement

The authors declare no potential or actual conflicts of interest.

Acknowledgements

This research was supported by National Institute on Aging(NIA) Contracts N01-AG-6-2101; N01-AG-6-2103; N01-AG-6-2106;NIA grants R01-AG028050 and R03-AG032498, NINR grant R01-NR012459; and Z01AG000951. This research was supported inpart by the Intramural Research Program of the NIH, NationalInstitute on Aging. The genome-wide association study was fundedby NIA grant 1R01AG032098-01A1 to Wake Forest UniversityHealth Sciences and genotyping services were provided by theCenter for Inherited Disease Research (CIDR). CIDR is fully fundedthrough a federal contract from the National Institutes of Health toJohns Hopkins University, contract number HHSN268200782096C.Data analyses for this study used the high-performance computa-tional capabilities of the Biowulf Linux cluster at the NationalInstitutes of Health, Bethesda, MD (http://biowulf.nih.gov).

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