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Neurobiology of Aging 33 (2012) 335–344

APOE �4 is associated with longer telomeres, and longer telomeresamong �4 carriers predicts worse episodic memory

Mikael Wikgrena,*, Thomas Karlssonb, Therese Nilbrinka, Katarina Nordfjällc,Johan Hultdind, Kristel Sleegerse,f, Christine Van Broeckhovene,f, Lars Nybergg,

Göran Roosc, Lars-Göran Nilssonh, Rolf Adolfssona, Karl-Fredrik Norrbacka

a Division of Psychiatry, Department of Clinical Sciences, Umeå University, SE-901 87 Umeå, Swedenb Department of Behavioral Sciences and Learning, Linköping University, SE-581 53 Linköping, Sweden

c Division of Pathology, Department of Medical Biosciences, Umeå University, SE-901 87 Umeå, Swedend Division of Clinical Chemistry, Department of Medical Biosciences, Umeå University, SE-901 87 Umeå, Sweden

e Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, Antwerp, Belgiumf University of Antwerp, BE-2610 Antwerp, Belgium

g Departments of Radiation Sciences (Radiology) and Integrative Medical Biology (Physiology), Umeå University, SE-901 87 Umeå, Swedenh Department of Psychology, Stockholm University, SE-106 91 Stockholm, Sweden

Received 6 November 2009; received in revised form 5 February 2010; accepted 3 March 2010

Abstract

Both leukocyte telomere length and the apolipoprotein �4 allele have been associated with mortality, cardiovascular disease, cognition,and dementia. The authors investigated whether leukocyte telomere length was associated with APOE genotype or cognitive abilities in theontext of APOE genotype. The setting for this cross-sectional study was 427 nondemented individuals aged 41–81 yr. The authors foundhat �4 carriers overall exhibited significantly longer telomeres compared with non-carriers (difference of 268 bp, p � 0.001). Thisifference was greatest at the lower limit of the age span and nonsignificant at the upper limit, which translated into a significantly higherelomere attrition rate (p � 0.049) among �4 carriers (37 bp/years) compared with non-carriers (21 bp/year). Further, longer telomeresmong �4 carriers significantly predicted worse performance on episodic memory tasks. No significant associations were found on tasksapping semantic and visuospatial ability, or among �3/�3 carriers. In conclusion, APOE �4 carriers had longer telomeres compared with

non-carriers, but higher rate of attrition. Among them, longer telomeres predicted worse performance on episodic memory tasks. Theseobservations suggest that the �4 allele is associated with abnormal cell turnover of functional and possibly clinical significance.© 2012 Elsevier Inc. All rights reserved.

Keywords: APOE; Cognition; Telomere length

www.elsevier.com/locate/neuaging

Telomeres are nucleoprotein structures forming the endsof eukaryotic chromosomes. They function as chromosomalcaps, protecting the free DNA ends from being subject ofrecombination or degradation events. In humans, telomeresare characterized by tandemly repeated TTAGGG se-quences, typically spanning between 4 and 10 kb in length.Due to an inherent incapability of the genomic replication

* Corresponding author at: Division of Psychiatry, Department of ClinicalSciences, Umeå University, SE-901 87 Umeå, Sweden. Tel.: �46703038942;ax: �4690135324.

E-mail address: mikael.wikgren@psychiat.umu.se (M. Wikgren).

0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.neurobiolaging.2010.03.004

machinery to replicate the full-length of the chromosome,telomeres shorten with each round of replication. If a crit-ically short telomere length (TL) is reached, the cell willapoptose or enter a state of replicative senescence (Allsoppet al., 1992; Harley et al., 1990; Zakian, 1995). Theseproperties suggest that TL may act as a biomarker forreplicative history and potential.

During the last decade, studies have found TL shorteningwith aging in several types of human tissues and cell typesin vivo, and the rate of shortening to be accelerated byoxidative stress and inflammatory states (Carrero et al.,

2008; Demissie et al., 2006; von Zglinicki, 2002). Epide-

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336 M. Wikgren et al. / Neurobiology of Aging 33 (2012) 335–344

miological studies have linked leukocyte TL with severalage-related diseases, among these Alzheimer’s disease, can-cer, cardiovascular disease, and all-cause mortality (Aviv etal., 2006; Bakaysa et al., 2007; Brouilette et al., 2007;Cawthon et al., 2003; Honig et al., 2006; Martin-Ruiz et al.,2006; Svenson and Roos, 2009; von Zglinicki et al., 2000).

One of the most widely studied genes is the apolipopro-tein E gene (APOE), a polymorphic gene with three majoralleles (�2, �3, and �4) coding for a protein with a centralrole in lipid metabolism and transport in tissue (Mahley,1988). In 1993, a landmark study found that the APOE �4,an allele carried by about a quarter of the population, wasassociated with a higher risk of developing Alzheimer’sdisease (AD) (Corder et al., 1993). Further, it has beenshown that the �4 allele negatively influences episodic, butnot semantic, memory and its age-related decline alsoamong nondemented individuals (Nilsson et al., 2006). Ad-ditionally, �4 carriers have elevated risks for cardiovasculardisease and impaired recovery following neurological insult(Bennet et al., 2007; Teasdale et al., 2005). APOE is also theonly gene demonstrated to meet all criteria for having pop-ulation level impact on mortality (Ewbank, 2004). The rel-atively rare APOE �2 allele is considered protective and themost common �3 allele neutral with respect to risk for theaforementioned conditions (Bennet et al., 2007; Corder etal., 1994).

The physiological underpinnings as to why dissimilarAPOE genotypes carry with them different risks are notcompletely understood. While the various APOE isoformsare associated with different levels of circulating lipids, it isrecognized that this only in part can explain the differencesin risks associated with each APOE genotype. Instead, re-cent research highlights allele-specific antioxidant abilitiesand modulatory effects on inflammation as possible explan-atory factors (Jofre-Monseny et al., 2008).

On the basis of the aforementioned findings of a coas-sociation of TL and APOE genotype with several disordersas well as the putative role of oxidative stress and inflam-mation in both TL dynamics (i.e., length and rate of short-ening) and APOE genotype-dependent pathogenicity, wehypothesized that telomere dynamics diverge between dif-ferent APOE genotypes. To investigate this hypothesis, wenalyzed 427 nondemented individuals representative of theeneral population, aged 41–81 yr, drawn from the Betulatudy in northern Sweden. Further, we wanted to explorehether TL has predictive power with respect to cognition

n the context of the APOE �4 allele in nondemented indi-iduals. For this, three cognitive domains were testedgainst TL in context of APOE genotype; episodic memory,emantic memory, and visuospatial abilities. Because agingypically is related to impaired performance in episodicemory tasks, and because effects of age are attenuated inany semantic memory tasks, we hypothesized that TLould be associated with episodic memory, leaving seman-

ic memory unaffected (or less strongly associated). Aging w

also brings about changes with respect to visuospatial abil-ities and because little is known about the roots to thiseffect, we deemed it instructive to clarify if TL or APOEenotype is related to visuospatial impairment.

. Methods

.1. Study participants

We studied a total of 427 individuals (205 males, 222emales; all Caucasian) aged 41–81 yr. All participantsere part of the larger Betula Study which has been de-

cribed in detail elsewhere (Nilsson et al., 1997, 2004). Inrief, the Betula Study is a prospective cohort study initiatedn 1988 aimed at exploring various aspects of memory,ealth, and aging. All participants were randomly selectedrom the population registry of Umeå, Sweden, and gavenformed consent. The study population has been validatedo conform well with the general population of northernweden. Exclusion criteria for enrollment into the Betularoject were dementia, mental retardation, serious visual oruditory handicaps, not having Swedish as a mother tongue,nd any other feature that would compromise the ability toomply with the study protocol. For details regarding theetula recruitment process, see Nilsson et al. (1997). From

he pool of individuals available, we initially included 440articipants, based on the aforementioned age-range. Six ofhese were excluded due to unsuccessful TL measurement.hree APOE genotype groups were formed; an �2 carrierroup (n � 49, comprised of two �2/�2 carriers and 472/�3 carriers), an �3/�3 group (n � 256), and an �4 carrierroup (n � 122, comprised of 101 �3/�4 carriers and 214/�4 carriers, including an oversampling of 10 �4 homozy-otes). Seven participants carrying the APOE �2/4 genotypeere excluded. This left us with a total group of 427 par-

icipants eligible for the present study. For the subgroupnalysis regarding associations between cognitive measuresnd TL, cognitive data were available on 173 participants.he 17 �2 carriers were due to their low numbers excluded

rom these analyses, leaving 93 �3/3 carriers and 63 �4arriers (52 �3/�4 carriers and 11 �4/�4 carriers).

The use of tobacco products was determined from mea-urements of plasma cotinine. Cotinine is the major metab-lite of nicotine, and measurement of it is the favoredethod of detecting use of tobacco products (Benowitz,

002). We measured cotinine in plasma samples obtainedrom participants and used 25 ng/mL as a cut-off for cate-orizing individuals as user or nonuser of tobacco products.he study was approved by the local ethics committee ofmeå University.

.2. Laboratory procedures

Leukocyte DNA was extracted from whole blood usingtandard procedures. APOE genotyping was performed asreviously described (Nilsson et al., 2006). Plasma cotinine

as measured by immunoassay (Immulite, 2000 nicotine

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337M. Wikgren et al. / Neurobiology of Aging 33 (2012) 335–344

metabolite assay; Siemens Medical Solutions Diagnostics,Los Angeles, CA). TLs were measured using a quantitativereal-time PCR-based technique described earlier (Cawthon,2002; Nordfjäll et al., 2007). In short, each DNA samplewas amplified on two parallel 96-well PCR plates; oneamplifying telomere repeats and the other amplifying asingle copy gene (�2-globin). The ratio of the mean telo-

ere repeat copy number to the mean single copy gene copyumber (T/S ratio) as related to that of a reference sampleDNA from the cell line CCRF-CEM) reflects relative TLs.elative TLs (measured in relative T/S ratios) were con-erted to telomere restriction fragment lengths (measured inase pairs) based on correlation data derived from sampleseasured using both quantitative real-time PCR and the

raditional Southern blot method.

.3. Cognitive assessments

Four tasks were designed to assess episodic memory.irst, participants were presented with two lists of shortentences that they were asked to memorize. Each list com-rised 16 sentences in imperative form; the sentences con-ained one verb and one noun. For one of the lists thearticipants were instructed to enact the sentence presentedo them. At the same time as the sentence was presented, anbject was provided; thus, participants were to carry out andemember “mini-tasks”. Following Cohen (1981), this pro-edure is designated subject performed task, SPT. The sec-nd list was also read to the participant. However, this timeo object was supplied and the participant listened to theentence. This verbal memory task is denominated VT.fter each list the subject was given a free recall test

immediate free recall, SPT and VT), during which partic-pants were asked to retrieve as many actions or sentencess possible. Later during the test session (approximately 30in after study) subjects were administered a recognition

ask. This task embodied 32 imperative sentences. Half ofhis corpus was not presented during study, the remainderccurred as SPTs or VTs during study. Participants weresked to indicate sentences that were presented duringtudy.

Two tests tapping semantic memory were used, the firsteing a revised version of the SRB vocabulary test (Dure-an and Sälde, 1971). Participants were presented with 30ords, accompanied by five words; one of which a synonym

o the target word. The subjects were instructed to indicatehich of the five words was a synonym. The second se-antic memory task was a word fluency test. The partici-

ant was instructed to generate as many words as possibleeginning with the letter “A” during 1 min.

In the block design test the subject was tasked to arrangeed and white blocks in the form of a pattern similar to thatf a target pattern. This visuospatial task is a subtest of theechsler Adult Intelligence Scale-Revised. Further details

egarding the cognitive tasks are available in Nilsson et al.

1997).

.4. Statistical analysis

One-way ANOVAs were used for testing differences ineans between continuous variables across APOE groups,

sing Brown-Forsythe tests to correct for unequal varianceetween groups where appropriate. To test for differences inategorical variables between APOE genotype groups wesed Pearson chi-square tests. Univariate ANCOVAs weresed to attain adjusted TLs (estimated marginal means) andelomere attrition rates; covariates included in the modelere age, gender, APOE genotype group, and an APOE*ge interaction term, with TL as the dependent variable.nadjusted correlation between age and TL was reported asearson correlation coefficient. When testing the predictiveower of TL regarding cognitive test scores, hierarchicalultiple regressions were employed with the cognitive test

core as the dependent variable. The first block of theegression included age, education, and gender as predictorariables (all entered simultaneously), in a second block, TLas introduced, thus permitting evaluation of its predictiveower above that offered by the variables in block one (byomparing r2 between the two blocks). We tested for addi-

tional potential confounding by individually including vari-ables (BMI, tobacco users, blood pressure, hypertension,cardiac disease, diabetes, prescription drugs) in the regres-sion models. Probability values below 0.05 were consideredsignificant. All statistical analyses were performed usingSPSS version 15.0.1 (SPSS, Inc., Chicago, IL).

2. Results

2.1. General characteristics of participants

The characteristics of our study population are shown inTable 1. The mean age of the 427 participants was 60.6 yr(ranging from 41–81) with males and females near equallyrepresented (48% males). For this study, the full sample wasdivided into groups according to the APOE genotype of theparticipants (see Table 1). BMI and regular use of tobaccoproducts and prescription medicines were similar acrossthese groups. Also, blood pressure and prevalence of hy-pertension, cardiac disease, and diabetes diagnoses did notdiffer significantly over the groups (Table 1).

One hundred fifty-six participants were eligible for anal-yses regarding cognitive measures. The characteristics ofthis slightly older subgroup (mean age � 67.1 yr; range45–81) are shown in Table 2. Similar to the full sample,there were no significant differences between the APOEgroups regarding the general characteristics (Table 2).

2.2. Telomere dynamics and APOE genotype

Leukocyte TL in the full sample averaged 5,544 bp inlength (range 3,737–9,803 bp), with a tendency of longerTL among females (difference of 112 bp, p � 0.121; ad-

justed for age). TL was significantly negatively correlated

Table 1General and telomere characteristics of the study participants

Full sample APOE group

Non-�4 carriers(�2/�2 � �2/�3 � �3/�3)

pnon-�4 v. �4

�2 carriers(�2/�2 � �2/�3)

p�2 v. �4

�3/�3 carriers p�3/�3 v. �2

�4 carriers(�3/�4 � �4/�4)

p�4 v. �3/�3

n 427 305 49 256 122Age, years (SD) 60.6 (9.9) 60.4 (10.1) 0.482 60.5 (10.6) 0.682 60.4 (10.0) 0.964 61.1 (9.5) 0.487Gender, male/female ratio

(% male)205/222 (48) 145/160 (48) 0.759 22/27 (45) 0.612 123/133 (48) 0.686 60/62 (49) 0.837

BMI, kg/m2 (SD) 26.2 (3.6) 26.3 (3.7) 0.136 26.3 (4.4) 0.571 26.3 (3.6) 0.884 25.9 (3.2) 0.282Tobacco users, n (%) 99 (23) 70 (23) 0.896 12 (25) 0.866 58 (23) 0.744 29 (24) 0.840Systolic blood pressure,

mm Hg (SD)138.1 (20.8) 138.0 (20.9) 0.879 140.4 (24.1) 0.559 137.5 (20.2) 0.378 138.3 (20.6) 0.725

Diastolic blood pressure,mm Hg (SD)

82.5 (9.6) 82.9 (9.7) 0.209 83.5 (9.4) 0.234 82.7 (9.8) 0.611 81.6 (9.1) 0.270

Hypertension, n (%) 204 (47.8) 142 (46.6) 0.426 27 (55.1) 0.612 115 (44.9) 0.191 62 (50.8) 0.283Cardiac disease, n (%) 57 (13) 40 (13) 0.822 7 (14) 0.952 33 (13) 0.791 17 (14) 0.779Diabetes, n (%) 19 (4) 14 (5) 0.824 1 (2) 0.509 13 (5) 0.352 5 (4) 0.676Prescription drugs (SD) 1.32 (1.7) 1.35 (1.7) 0.570 1.73 (1.9) 0.093 1.28 (1.7) 0.089 1.25 (1.8) 0.868Telomere length, bp (SD) 5544 (787) 5475 (696) 0.012 5514 (794) 0.127 5467 (677) 0.697 5716 (959) 0.011Telomere length,

adjusted, bp (SE)a5567 (44) 5467 (42) 0.001 5507 (105) 0.068 5459 (46) 0.676 5735 (67) 0.001

Telomere attrition rate,bp/year (SE)b

25 (4) 21 (8) 0.049 8 (12) 0.017 24 (8) 0.151 37 (7) 0.113

Telomere length at age41, bp (SE)c

6018 (96) 5879 (91) 0.001 5664 (220) 0.003 5926 (100) 0.280 6463 (157) 0.004

Telomere length at age61, bp (SE)

5558 (44) 5459 (42) 0.001 5504 (105) 0.083 5450 (46) 0.637 5720 (67) 0.001

Telomere length at age81, bp (SE)

5099 (99) 5039 (96) 0.736 5345 (230) 0.187 4974 (105) 0.145 4978 (155) 0.986

All values are means unless otherwise stated. SD, standard deviation; SE, standard error.a Adjusted for age and gender.b Adjusted for gender.c Estimated marginal means, evaluated at given ages, adjusted for gender.

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339M. Wikgren et al. / Neurobiology of Aging 33 (2012) 335–344

with age (r � �0.315; p � 0.001), with an average attritionrate of 25 bp/year (Table 1).

Comparing TL between the three APOE groups showedthat the �4 allele is associated with relatively longer telo-meres (Table 1). There was a significant difference of 249bp (p � 0.011) between �4 carriers and �3 homozygotes,which, after adjusting for age and gender, increased to 276bp (p � 0.001). The adjusted difference between �4 and �2arriers was 228 bp (p � 0.068). There were no significantifferences in TL between the �2 and �3/�3 carriers (p �

0.697; adjusted for age and gender). Overall, comparingcarriers of the �4 allele with non-carriers, i.e., �2 and �3/�3arriers (n � 305), showed that �4 carriers had significantlyonger TL; the adjusted difference being 268 bp (p �.001).

The TL data from the smaller subgroup is analogous tohat of the full sample, with an adjusted TL difference of58 bp (p � 0.004) between �3/�3 and �4 carriers. The TLs

in these two APOE groups were slightly shorter compared

Table 2General, telomere and cognitive characteristics of cognitive analysissubgroup

APOE group

�3/�3 carriers �4 carriers p

93 63eneral and telomere characteristicsAge, years (SD) 67.8 (6.9) 66.2 (7.9) 0.188Gender, male/female ratio

(% male)45/48 (48) 27/36 (43) 0.497

Education, years (SD) 9.9 (3.7) 9.7 (3.6) 0.759BMI, kg/m2 (SD) 26.8 (3.5) 26.3 (3.3) 0.372Tobacco users, n (%) 11 (12) 9 (14) 0.652Systolic blood pressure, mm Hg

(SD)147.9 (18.6) 147.3 (17.8) 0.883

Diastolic blood pressure, mmHg (SD)

82.1 (9.4) 82.1 (9.8) 0.985

Hypertension, n (%) 59 (63) 45 (71) 0.299Cardiac disease, n (%) 18 (19) 11 (17) 0.765Diabetes, n (%) 6 (6) 5 (8) 0.722Prescription drugs (SD) 1.38 (1.8) 1.30 (1.4) 0.784Telomere length, bp (SD) 5348 (499) 5641 (638) 0.002Telomere length, adjusted, bp

(SE)a5350 (57) 5608 (69) 0.004

ognitive domain and taskpisodic memoryImmediate Free Recall, SPT (SD) 8.15 (2.92) 8.17 (2.85) 0.959Immediate Free Recall, VT (SD) 4.84 (2.46) 5.07 (2.44)c 0.575Delayed recognition, SPTb (SD) 6.90 (1.02) 6.43 (1.68) 0.048Delayed recognition, VTb (SD) 5.58 (1.91) 4.90 (1.92) 0.032

Semantic memory and languageVerbal fluency (SD) 10.41 (4.25) 11.52 (4.44) 0.116SRB vocabulary (SD) 22.67 (4.41) 23.52 (4.37) 0.234

Visuospatial functionsBlock design (SD) 26.12 (8.46) 26.62 (9.27) 0.728

All values are means unless stated otherwise. SD, standard deviation; SE,standard error.

a adjusted for age and gender.b denotes hits minus false alarms.c n � 61.

ith corresponding groups in the full sample, but this could

be expected considering the slightly higher mean age of theparticipants in these smaller subgroups and the establishedTL attrition with advancing age.

Analysis of TL as a function of age, i.e., attrition rate(based on cross-sectional data), revealed inequalities be-tween the three APOE groups (Fig. 1). Among �2 carriers,TL diminished very little with increasing age (8 bp/year).The �4 carriers had a significantly higher rate (37 bp/year)compared with the �2 carriers (p � 0.017). In this context,carriers of the �3/�3 genotype exhibited a medium rate ofattrition (24 bp/year). Non-�4 carriers had a significantlylower rate of attrition (21 bp/year) compared with �4 car-riers (p � 0.049). Consequently, differences in TL betweenAPOE groups varied depending on age, which is illustratedby the estimated marginal means (Table 1). The differencein TL between �4 and �3/�3 carriers was at its greatest, 537bp (p � 0.004), at the beginning of the age-span in ourcohort (age 41), while there was no significant difference(p � 0.986) at the upper age limit. With diminishing differ-ences with increasing age, the difference in TL between �4and �3/�3 carriers ceased to be significant at age 68 (withthe adjusted difference at age 68 being 177 bp; p � 0.079).The corresponding cut-off point for carriers vs. non-carriersof the �4 allele was 67 yr. At age 81, the �2 group exhibitedthe longest TL, albeit it did not reach significance (p �0.145; compared with �3/�3 carriers).

Additional adjustments for any of the potential con-founders (BMI, tobacco usage, blood pressure, hyperten-

Fig. 1. Telomere attrition rate as a function of APOE group. Shown aremean attrition rates, adjusted for gender, with error bars representingstandard error. Not represented in the figure are non-�4 carriers (i.e., �2/�2,�2/�3 and �3/�3 carriers grouped together), which had an attrition rate (21bp/year, SE � 8) significantly different from that of �4 carriers (p �

0.049).

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340 M. Wikgren et al. / Neurobiology of Aging 33 (2012) 335–344

sion, cardiac disease, diabetes, prescription drugs usage)did not alter the significance of our results reportedabove.

2.3. Telomeres and cognition

Performance on tasks drawing on cognitive resources ispresented in Table 2. As expected, �4 carriers performedworse than �3 homozygotes on delayed (episodic) memorytasks but not on semantic memory and visuospatial tasks.

To test the ability of TL to predict cognitive perfor-mance, we carried out a series of hierarchical regressionanalyses. In these analyses, results pertaining to cognitivemeasures were regressed upon TL and variables known toinfluence cognition in two steps; the first step comprisingbackground information (i.e., age, gender, and education)and the second step adding TL to the model. Separateanalyses were conducted for participants belonging to theAPOE �3/�3 genotype and participants displaying the

POE �3/�4 or �4/�4 genotypes.The results of the hierarchical regression analyses are

hown in Tables 3 and 4. Statistically significant TL effectsere noted with respect to �4 carriers regarding two epi-

odic memory tasks; immediate free recall of SPTs (� ��0.26; p � 0.026) and delayed recognition of SPTs (� �

0.31; p � 0.016). In the model concerning immediate freeecall of SPTs among �4 carriers, the addition of TL in theecond step of the regression provided an additional 6%

Table 3Hierarchical regression examining telomere length as predictor of episodi

Group step and predictor variable APOE �3/�3

� p

mmediate Free Recall SPTirst stepAge �0.37 � 0.001Education 0.20 0.042Gender 0.04 0.679

Second stepTelomere length 0.00 0.971

Immediate Free Recall VTFirst step

Age �0.18 0.075Education 0.31 0.003Gender �0.02 0.862

Second stepTelomere length �0.10 0.327

Delayed recognition SPTFirst step

Age �0.32 0.004Education �0.05 0.633Gender �0.01 0.949

Second stepTelomere length �0.19 0.072

Delayed recognition VTFirst step

Age 0.07 0.530Education 0.28 0.012Gender 0.01 0.921

Second step

Telomere length �0.09 0.401

p � 0.026) of variance explanation (r2), amounting to a totalof 35% (p � 0.001). Regarding delayed recognition of SPTsamong �4 carriers, TL added 9% (p � 0.016) of varianceexplanation to the 12% explained by age, gender, and edu-cation, totaling 21% of variance explanation (p � 0.009).No significant contributions by TL was noted among APOE�3/�3 carriers regarding SPTs (although it should be notedthat the association was marginally significant pertaining toSPT delayed recognition, � � �0.19; p � 0.072) or VTsTable 3). Concerning semantic tasks (i.e., verbal fluencynd vocabulary tests); TL did not significantly predict per-ormance in any of the APOE groups. Education stood outs significantly associated with semantic performance, withge having no influence over performance. In the visuospa-ial block design test, age and education were the mostnfluential variables in both groups (Table 4), with TLaving no significant effect. In summary, longer TL ad-usted for age, gender, and education within the group of �4arriers predicted worse scores on both episodic SPTs. Theattern among �4 carriers was that strong associations be-ween age and a cognitive dependent variable was coinci-ent with significant associations between TL and the sameognitive outcome variable (being episodic tasks), with ex-eption for the block design variable.

Additional adjustments (for BMI, tobacco usage, bloodressure, cardiac disease, diabetes, or drug usage) did notlter any of the above reported significant results.

ry abilities

APOE �4

p � p r2 p

� 0.001 0.29 � 0.001�0.40 0.002

0.10 0.4150.28 0.014

� 0.001 0.35 � 0.001�0.26 0.026

0.001 0.11 0.077�0.16 0.238

0.23 0.0950.00 0.976

0.002 0.12 0.1370.06 0.668

0.032 0.12 0.054�0.27 0.054�0.03 0.984

0.21 0.0930.017 0.21 0.009

�0.31 0.016

0.096 0.03 0.7080.00 0.990.11 0.4580.12 0.378

0.134 0.03 0.846

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341M. Wikgren et al. / Neurobiology of Aging 33 (2012) 335–344

3. Discussion

The first main finding was that carriers of the APOE �4llele overall had significantly longer leukocyte TL com-ared with non-�4 carriers and that TL attrition rate wasenotype dependent with �4, �3/�3, and �2 carriers losing

37, 24, and 8 bp/year, respectively. Hence, it follows thatthe differences in TL between APOE genotypes variedacross the age span. The difference between �4 carriers andnon-carriers was most pronounced at the lower limit of theage span (584 bp), 41 yr. With increasing age, the differ-ences diminished, reaching a nonsignificant difference atage 67, and further decreasing to a negligible difference atthe upper age limit of 81 yr. Within the full sample, theyearly decline in TL was 25 bp which is in good accordancewith that reported by others (Aviv et al., 2006; Demissie etal., 2006).

To the best of our knowledge, only four prior studieshave addressed a possible association between APOE geno-type and TL. Only Starr et al. (2008) reported significantfindings; they found that TL was significantly shorteramong �4 carriers in a sample of 190 individuals (undis-closed number of �4 carriers), all 79 yr of age. In our studypopulation, there were no significant differences in TL be-tween different APOE genotype carriers at the age of 79; TLamong �4 carriers, compared with non-carriers, was signif-icantly longer at age 66 and below. The other three studiesreported no significant findings (Honig et al., 2006; Martin-Ruiz et al., 2006; von Zglinicki et al., 2000). However, thesestudies differ from our investigation in some noteworthy

Table 4Hierarchical regression examining telomere length as predictor of semant

Group step and predictor variable APOE �3/�3

� p

Semantic memory and languageVerbal fluencyFirst step

Age �0.08 0.429Education 0.39 � 0.001Gender �0.04 0.701

Second stepTelomere length 0.00 0.974

SRB vocabularyFirst step

Age �0.03 0.730Education 0.48 � 0.001Gender �0.11 0.249

Second stepTelomere length �0.09 0.352

Visuospatial functionsBlock designFirst step

Age �0.28 0.008Education 0.18 0.079Gender �0.20 0.044

Second stepTelomere length 0.05 0.626

respects. Samples were mixed (patients and healthy indi- t

viduals) and smaller compared with the sample we studied.Also, two (Honig et al., 2006; Martin-Ruiz et al., 2006)studied samples with an average age of over 80 yr, an agewhere we did not find significant differences between thedifferent genotypes (the third study, by von Zglinicki et al.(2000), did not report the age of the subsample used forinvestigating potential differences in TL between APOEenotypes).

The second main finding was that among �4 carriers,onger TL predicted worse performance on two episodicemory tasks (the two SPT tasks). No association was

ound between TL and semantic or visuospatial skills. Outf these three principal cognitive modalities being tested,pisodic memory is the one most tightly connected with theging process and thus the one most likely to be associatedith TL. Out of the seven cognitive scores, age was a

ignificant predictor only in the two episodic SPT tests andn the visuospatial block design test. Thus, the emergingattern was that of a coinciding significant prediction by TLnd age of two episodic tasks, but present only among the4 carriers (block design being the exception because ageut not TL was a significant predictor for this visuospatialask). Also, the �4 carriers exhibited significantly lowercores on episodic memory (SPT and VT recognition) com-ared with �3/�3 participants, which is consistent with pre-ious findings (Nilsson et al., 2006).

Our findings could be contrasted with two earlier reportsn the relationship between TL and cognition. Harris et al.2006) examined 185 individuals who were all 79 yr old. In

isuospatial abilities

APOE �4

p � p r2 p

0.001 0.18 0.009�0.04 0.750

0.40 0.003�0.00 0.974

0.002 0.22 0.006�0.21 0.087

� 0.001 0.18 0.008�0.03 0.840

0.41 0.003�0.02 0.881

� 0.001 0.18 0.0190.05 0.704

0.001 0.28 � 0.001�0.33 0.011

0.29 0.022�0.09 0.415

0.002 0.28 0.0010.02 0.893

ic and v

r2

0.18

0.18

0.25

0.26

0.17

0.18

his older sample, there was no association between TL and

fttdwwc

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342 M. Wikgren et al. / Neurobiology of Aging 33 (2012) 335–344

cognition. Recently, Valdes et al. (2008) examined 382emale twins, aged 19–78 years, using computerized taskshat tapped reaction time and working memory. Correctinghe results for an estimate of prior intellectual capacity, TLisplayed a weak, but statistically significant correlationith some of the cognitive measures. However, in contrastith our data, Valdes et al. (2008) documented a positive

orrelation between TL and cognition.There are two distinct differences between our study and

he reports by Harris et al. (2006) and Valdes et al. (2008).First, our sample was stratified with respect to APOE ge-otype. Given that our observation of an association be-ween TL and memory was primarily among the �4 carriers,he unknown distribution of APOE genotypes in the previ-

ous studies could have influenced the results. A seconddifference lies in the choice of cognitive measures. Ourinvestigation comprised tasks and measures that are highlysensitive to the effects of aging and brain damage. In par-ticular, we emphasized performance in episodic memorytasks; whereas the study by Valdes et al. (2008) highlightedtimed performance in tasks of relatively brief duration.Although such tasks may be highly relevant when it comesto the detection of the parietal and frontal lesions as seen indementia, nondemented elderly typically have more pro-nounced problems in tasks that draw on the integrity of themedial temporal lobe, that is, episodic tasks (Park andReuter-Lorenz, 2009; Persson et al., 2006).

The APOE �4 allele is an established risk factor forcardiovascular disease and dementia (Bennet et al., 2007;Corder et al., 1993); diagnoses which also have been linkedwith relatively short TL (Brouilette et al., 2007; Martin-Ruiz et al., 2006; von Zglinicki et al., 2000). The findingthat APOE �4 carriers exhibited longer TL was thereforesurprising. However, most studies linking TL with cardio-vascular disease and dementia have not considered theAPOE genotype of their study participants (the typical fre-quency of the �4 allele in the general population is 15–25%)(Bennet et al., 2007). Another aspect to consider is that welooked exclusively at a representative population sample ofnondemented individuals. Telomere dynamics in diseased�4 carriers are not necessarily similar to those observed inhealthy �4 carriers. It is possible that the higher telomereattrition rate seen in the �4 carrier group is a reflection of theincreased risk of disease associated with the gene variant,with diseased �4 carriers potentially having even greaterattrition rates leading to shorter TL compared with healthy�4 carriers and non-�4 carriers.

Few epidemiological studies have addressed telomereattrition rate. One reason for this is the more resource-demanding task of procuring the longitudinal data needed toexplore intraindividual telomeric attrition rates. The alter-native is to derive an attrition rate from cross-sectional datawith TL measured on subjects of different age. In onelongitudinal study, a higher TL attrition rate was found to be

associated with a rise in insulin resistance (Gardner et al.,

2005), and in a recent study by Guan et al. (2008) TLattrition rate, but not TL, was higher among Parkinson’sdisease patients compared with healthy controls.

To a certain degree, telomere attrition rate is proportionalto the length of telomeres, recently shown by us (Nordfjället al., 2009) and Aviv et al. (2009) in longitudinal TL data.In addition to TL, oxidative stress is recognized as animportant modulator of telomeric attrition rate, with higherlevels of oxidative stress leading to higher degrees of attri-tion (von Zglinicki, 2002). Also, chronic inflammation in-creases replication of hematopoietic stem cells as they needto compensate for the higher turnover rates of circulatingleukocytes; a process leading to increased telomere attritionrate in hematopoietic stem cells and consequently shorterTL in circulating leukocytes. Recently, evidence has beenbuilding up implicating oxidative stress and inflammationas contributing to the different degrees of disease vulner-ability conferred by different APOE genotypes. The �4allele has been linked to a proinflammatory state, and thethree apoE isoforms shown to possess different levels ofantioxidant properties (�2 � �3 � �4) (Jofre-Monseny etal., 2008). This predicts that different setups of APOEalleles impact TL dynamics differently; a predictionwhich fits with the observation of genotype-specific at-trition rates (�2 � �3 � �4).

Based solely on the observation of a higher attrition rateand evidence suggesting higher oxidative burden among �4carriers, one would expect relatively short TLs among �4carriers. A readily available explanation accounting for thedifference in TL, suggested to originate somewhere in theage-span below 41 yr, is not available. One possible expla-nation is that the �4 allele via unknown mechanisms conferssome antiproliferative attributes, thus conserving TL. Suchan effect could occur during fetal development and inyounger years generate the TL differences observed. Withincreasing age and thus increasing burden of oxidativestress and chronic inflammation, �4 carriers (Kregel andZhang, 2007), being more vulnerable to both, would seerelatively higher increases in rates of attrition and thusdiminishing TL differences. This hypothesis not only ac-counts for why �4 carriers have longer TL, but also offers anexplanation as to why we observe an inverse relationshipbetween TL and episodic memory among �4 carriers.

The dentate gyrus in the hippocampus, a key region forthe functioning of episodic memory, is one of the few areasin the adult brain exhibiting neurogenesis (Eriksson et al.,1998). This process is thought to be important for theformation of new memories, and as such an inhibition ofneurogenesis in the hippocampus can be expected to, withsome degree, inhibit functions related to this brain structure(Winocur et al., 2006). This, together with the hypothesizedanti-proliferatory effects (affecting both hippocampal neu-rogenesis and the immune system) in �4 carriers, may ex-plain the observed positive covariance between leukocyte

TL and episodic memory among �4 carriers.

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343M. Wikgren et al. / Neurobiology of Aging 33 (2012) 335–344

Also, imaging studies have shown that the hippocampusnd dentate gyrus of nondemented �4 carriers typically aremaller compared with those of non-carriers (Lind et al., 2006;ueller et al., 2008), compatible with there being a factor

ounteracting neurogenesis. If this is an early-onset atrophy ofhe hippocampus among individuals progressing towards de-entia, or if �4 carriers have smaller hippocampal volumes

lready at birth or early childhood is not known.Although we measured TL in circulating leukocytes,

tudies indicate that TL in different tissues correlate intra-ndividually, i.e., an individual having relatively long TL ineukocytes typically has relatively long TL in other tissuess well (Friedrich et al., 2000). This suggests that our �4arriers with relatively long leukocyte TL have relativelyong TL in hippocampal tissue as well. Interestingly,homas et al. (2008) found that individuals suffering fromD on average had longer TL in hippocampal tissue, com-ared with healthy controls. Because TL to some extent isn indicator of proliferative history, this suggests a historyf relatively low proliferatory activity in the hippocampusf AD patients. In view of our findings and the fact that the4 allele is overrepresented among AD patients, it wasnfortunate that Thomas et al. did not identify APOE statusf the subjects studied.

Our results spawn questions whose answers are not pos-ible to extract from the current study. Extrapolation of theata predicts even greater differences in TL between �4 andon-�4 carriers at ages below the age-limit of our studyopulation, and above the age-limit, �4 carriers are pre-icted to have shorter TL. The study of younger and olderohorts are needed to explore the relationship between TLnd APOE outside the age-limits of the present study andore studies, preferentially longitudinal, are warranted to

onfirm our findings and further explore the relationshipsnd hypotheses postulated here. The results may indicate aovel pathophysiological factor associated with the APOE4 allele; namely inhibition of cell proliferation. Also, highL attrition rate among immune cells may late in life lead to

elomere exhaustion, resulting in replicative immunosenes-ence. Combining APOE status with telomere dynamicsnformation might emerge as a combined clinically usefulredictor of cognitive decline and other APOE related dis-ase states. Our study also highlights APOE polymorphisms a possible source of confounding in prior telomere lengthtudies, particularly so in studies involving disorders asso-iated with APOE genotype.

isclosure statement

The authors have no conflicts of interest.

cknowledgments

The research was funded by the Swedish Research Coun-

il (grant 2,006–4,472), the Medical Faculty, Umeå Uni-

ersity, and the County Councils of Västerbotten andorrbotten, Sweden. The Betula Study is supported byrants 345-2003-3883 and 315-2004-6977 from the Swed-

sh Research Council. The APOE genotyping was funded byhe Fund for Scientific Research – Flanders (FWO-V). K S.s holder of a postdoctoral fellowship of the FWO-V. Re-earch nurses Annelie Nordin and Eva Lundberg are thank-ully acknowledged for their help and expertise. We alsocknowledge the statistical expertise of Birgitta TörnkvistDepartment of Statistics, Umeå University).

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