Volumes of hippocampus, amygdala and frontal lobe in Alzheimer patients with different apolipoprotein E genotypes

Neuroscience Vol. 67, No. 1, pp. 65-72, 1995

~ ) Pergamon

0306-4522(95)00014-3

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VOLUMES OF HIPPOCAMPUS, A M Y G D A L A A N D FRONTAL LOBE IN ALZHEIMER PATIENTS WITH DIFFERENT APOLIPOPROTEIN E GENOTYPES M. LEHTOVIRTA,* M. P. LAAKSO,* H. SOININEN,*rr S. HELISALMI,t A. M A N N E R M A A , t E.-L. HELKALA,* K. PARTANEN,:~ M. RYYN)~NEN,t P. VAINIO,~ P. HARTIKAINEN* and P. J. R I E K K I N E N Sr§ *Department of Neurology, Unit of Clinical Genetics of the tDepartment of Gynecology and Obstetrics, ~Department of Radiology, Kuopio University Hospital, University of Kuopio, §A. I. Virtanen Institute, P.O. Box 1627 Kuopio, Finland Abstract--An increased frequency of apolipoprotein E E4 allele has been reported in patients with late onset Alzheimer's disease. Apolipoprotein E participates in the transport of cholesterol and other lipids and interferes with the growth and regeneration of both peripheral and central nervous system tissues during development and after injury. Apolipoprotein E is also implicated in synaptogenesis. Apolipoprotein E isoforms differ in binding to amyloid-fl-protein and tau protein in vitro. Here, we wanted to study the effect of apolipoprotein E genotype on the magnitude of damage in the hippocampus, where a marked synapse loss exists in Alzheimer's disease. We measured by magnetic resonance imaging the volumes of the hippocampus, amygdala, and frontal lobes in the three Alzheimer subgroups: patients with 2, 1 or 0 FA alleles. We also investigated the profile of deficits on tests assessing memory, language, visuospatial, executive, and praxic functions of these Alzheimer subgroups. All Alzheimer patients were at early stage of the disease. We found that Alzheimer patients with E4/4 genotype (N = 5) had smaller volumes of the hippocampus and the amygdala than those with E3/4 (N = 9) and those with E3/3 or E2/3 (N = 12). The difference was significant for the right hippocampus (-54% of control) and the right amygdala (-37% of control). The volumes of the frontal lobes were similar across the Alzheimer subgroups. The patients with E4/4 also showed lowest scores on delayed memory tests and differed from E3/3, 3/2 patients in the list learning test (<0.05). The Alzheimer subgroups did not differ in age, age at onset, the global clinical severity of dementia or in the profile of other neuropsychological deficits. The results suggest that Alzheimer patients with apolipoprotein E E4/4 genotype have severe damage of the hippocampus and amygdala very early in the disease process and differ from Alzheimer patients with one or no E4 allele. Whether this larger volume loss in the hippocampus is related to increased accumulation of the amyloid and paired helical filaments or greater loss of synapses in E4 homozygous Alzheimer patients, is of interest and worth studying.

Several studies have reported an increased frequency of apolipoprotein E (ApoE) allele E4 among patients with late onset familial and sporadic Alzheimer's disease (AD). 3°'35'42,49In late onset families, risk of AD increased from 20 to 90% and the mean age of onset decreased with increasing number of ApoE E4 alleles: An earlier study suggested linkage of late onset familial AD to the proximal long arm of chromosome 19 in the region where the ApoE gene is localized. 32 ApoE is a plasma protein that binds to low-density lipoprotein receptor and is involved in the transport of cholesterol and other lipids in various cells of the body. 21 The alleles E2, E3 and E4 determine ApoE polymorphism and result in six phenotypes E2/2, E2/3, E2/4, E3/3, E3/4 and E4/4. The subjects with

IITo whom correspondence should be addressed. Abbreviations: AD, Alzheimer's disease; ApoE, apolipo-

protein E; EDTA, ethylene diaminetetra-acetate; MRI, magentic resonance imaging; MMSE, mini-mental status examination. 65

E4 allele have higher levels of total and low-densitylipoprotein cholesterol 52 and a higher risk for myocardial infarction and coronary heart disease25 than those with ApoE 3/3. Apolipoprotein E also has relevance to the nervous system. It is implicated in the growth and regeneration of both peripheral and central nervous tissues during development and after various types of injury. In the central nervous system astrocytes synthesize ApoE. Injury of brain tissue induces considerable increase of ApoE mRNA in the astrocytes. 33 Immunohistochemical studies have demonstrated the presence of ApoE in senile plaques, neurofibrillary tangles, and cerebrovascular amyloid. 2s'56The in vitro binding of ApoE of the cerebrospinal fluid to synthetic amyloid-fl-protein and differential binding of ApoE isoforms to tau protein suggest that ApoE might be involved in the pathogenesis of AD. 49'5° Alzheimer's diesease is a heterogenous entity. 23The disease most commonly presents with a loss of memory followed by dysfunctions in visuospatial skills,

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M. Lehtovirta et al.

executive functions, a n d language. Recent m a g n e t i c resonance imaging ( M R I ) studies have indicated a p r o n o u n c e d decline in volumes o f the h i p p o c a m p u s as a n early sign o f A D . L7'19'2°'46 F u r t h e r m o r e , amygdaloid volumes decrease relatively early in the disease process. 5'45 A t t e n u a t e d plastic response a n d loss o f synapses in the neocortex a n d the h i p p o c a m p u s have been reported in A D brain. 16'22'37 Experimental studies have suggested t h a t A p o E m a y be implicated in the synaptogenesis o f the h i p p o c a m p u s . 34 In the present study, we w a n t e d to investigate whether patients with different A p o E genotypes differ in the degree of the d a m a g e in the h i p p o c a m p u s a n d the amygdala, structures k n o w n to be affected early in A D . The hypothesis we a t t e m p t e d to test, was t h a t A D patients carrying A p o E E4 allele will have more p r o n o u n c e d atrophy, particularly, in the hippocampus, where synaptogenesis is strong d u r i n g m e m o r y processing. ~ C o n c u r r e n t l y with p r o m i n e n t h i p p o c a m p a l atrophy, we expected to find more severe m e m o r y i m p a i r m e n t in A D patients with A p o E E4 allele t h a n in those without. T o this end, we m e a s u r e d by M R I the volumes of the hippocampus, a m y g d a l a a n d frontal lobes in the A D patients with A p o E E4/4, E3/4 a n d E3/3 or 3/2 genotypes d e t e r m i n e d by using PCR-restriction enzyme m e t h o d . The m e a s u r e m e n t o f the frontal lobe was included to represent the neocortical i n v o l v e m e n t of the A D brain. We also e x a m i n e d the profile of deficits o n tests assessing m e m o r y a n d o t h e r cognitive functions such as language, visuospatial, executive a n d praxic functions of these A D subgroups. EXPERIMENTAL PROCEDURE

Patients

We studied 26 patients fulfilling the criteria of probable AD 24 defined by the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association work group and 16 ageand sex-matched healthy cognitively normal controls. Table 1 presents the clinical characteristics of the subjects. The local ethics committee approved the study. All subjects and also caregivers of demented patients gave informed consent for the participation in the study.

The AD patients were either at diagnostic examinations or recently diagnosed. The mean duration of the disease was 28 _+ 18 months. Fourteen had late onset (/>65 years) and 12 had early onset (<65 years) of the disease. The patients underwent the following examinations: general physical and clinical neurological examination; assessment of clinical severity using mini-mental status examination (MMSE); m assessment of extrapyramidal signs using the Webster Parkinson's disease scale; 54assessment of depressive signs by the Hamilton scale; ~3 extensive battery of laboratory tests to exclude secondary causes of dementia; neuropsychological tests; electroencephalogram and event-related evoked potentials; magnetic resonance imaging of the brain. The controls underwent the same protocol except Webster and Hamilton scales. 47 All subjects had a score of less than four in the modified ischemic scale, as Neuropsychological tests

Memory functions were examined with list learning test using shopping items. ~4 A "yes" or "no" recognition of the words in the list was asked after a 30-min delay filled with other psychometric tests. Immediate memory was also examined with the Heaton visual reproduction test. 4° The delayed recall of the figures was asked after a 30-min delay filled with other psychometric tests. Verbal functions were examined with the Boston naming test. TM Visuospatial functions were examined with copy a cube test, clock setting test and the block design subtest of the Wechsler adult intelligence scale. "'55 Praxic functions of the hand were investigated using Luria's method, choosing three examples of each functional area: simple movements, kinesthetic basis of movement, visuospatial organization, dynamic organization of motor act, verbal regulation of motor act, ideomotor praxia. The maximum score of praxic functions was 48. ~4 To assess executive functions we applied the Wisconsin card sorting test using Nelson's version,29 trail-making tests A and B, 36 and verbal fluency. 2 The maximum time of 150 seconds for trail-making A and 300 s of trail making B was allowed. If the test was not completed in time allowed, the missing letters or numbers were scored as omissions. In the verbal fluency test the subject was asked to produce as many words as they could beginning with letters P, A and S in 1 min for each letter. The score was the number of words correctly named. Magnetic resonance imaging technique

The subjects were scanned with a 1.5T Magnetom (Siemens, Erlangen) by using the standard head coil and a tilted coronal 3D gradient echo sequence (MP-RAGE: TR 10ms, TE 4ms, TI 250ms, flip angle 12°, FOV 250mm, matrix 256 x 192, one acquisition). This resulted in 128 Tl-weighted partitions with slice thickness of 1.5-1.8 mm oriented at right angle to the long axis of the hippocampus.

Table 1. Clinical characteristics of controls and Alzheimer patients of different apolipoprotein E genotypes

Controls (N = 16) Women/men Age, years Age at onset, years Education (years) Mini-mental status Webster Hamilton

10/6 70.2+4.7 -11.3 ___3.0* 28.6 __+1.4" ---

Alzheimer patients Apolipoprotein E genotype 4/4 3/4 3/3 or 3/2 (N = 5) (N = 9) (N = 12) 3/2 65.0_+ 11.4 62.2 + 11.3 6.8 + 1.8 22.6 +_ 3.0 2.6 + 3.4 4.0 + 4.6

4/5 71.6_+7.6 70.0 _+ 8.2 7.5 _+ 6.2 21,1 ___4.6 4.1 _+2.7 4.8 _+ 2.4

5/7 68.5+8.7 66.2 +__9.0 5.5 ___1.3 23.6 + 3.6 1.9 + 2.9 2.4 + 3.3

Results are expressed as mean ___S.D. ANOVA ove the study groups (P < 0.0001) show significant difference in education and mini-mental status; Duncan, controls versus Alzheimer subgroups, *P < 0.05.

Hippocampus, apolip0protein E and Alzheimer's disease The radiologist who analysed the MRIs was blinded to the subject's clinical data. The method has been described earlier in detail. 47

Determination of volumes of the hippocampus and the amygdala. We used standard anatomical atlases of the human brain 6'8 with some adjustment from previous literature 53 as guidelines to determine the boundaries of the amygdala and the hippocampus in oblique coronal MRI sections.47 The outlining of the hippocampus and the amygdala has been previously reported in detail, a7 The boundaries of the region of interest were outlined by a tractball driven cursor proceeding from anterior to posterior. The number of voxels within the region was calculated by using an in-house developed program for standard work console. Terminology of anatomical regions studied. In this study, the outlines of the amgydala included the deep nuclei of the amygdala, the superficial nuclei of the amygdala, and the remaining nuclei of the amygdala. 47 At the most rostral sections of the amygdala, we outlined only the deep amygdaloid nuclei in the MRI image to avoid the overestimation of the amygdaloid volume due to inclusion of the piriform cortex. This, on the other hand, may have resulted in exclusion of the most rostral portions of the periamygdaloid cortex from the total amygdaloid volume. The hippocampus included the dentate gyrus, the hippocampus proper and the subicular complex. The uncal portion of the rostral hippocampus that is located ventral to the caudal amygdala was included into the hippocampus. The caudal end of the hippocampus was determined from the section, in which the fornices were still detectable in their full length. Determination of volumes of frontal lobes. The gyri were manually outlined on every third slice separately on the right and the left hemisphere. Thus, due to thin slices, the measurement was done at an interval of 5 mm. The most anterior of the slices was the one with clearly visible gyri. On the most posterior slices, a straight line was drawn from the bottom of the lateral fissure to the choroidal fissure in order to separate the temporal lobe from the frontal lobe. From the bottom of the choroidal fissure a line was drawn above the optic tract to the midline and then a line was drawn vertically to the interhemispheric cerebral fissure. The most caudal slice included in the measurement was the one in which the anterior commissure was present. The volume of the lateral ventricles was also measured and consequently subtracted from the volume of the slice. The volume of each slice was multiplied by three and thereafter, the slice volumes were summed up. Normalization of volumes. Normalization was done by dividing the volumes of the hippocampus, amygdala, or the frontal lobe by brain area. To obtain brain area, we measured the area of both hemispheres in a M R image taken at the level of the anterior commissure. The normalization yielded ratios: volume of the region of interest/brain area. We used normalized values in all statistical analyses.

Determination of apolipoprotein E genotype Genomic DNA extraction and polymerase chain reaction amplification. Samples of 10 ml venous blood were collected in EDTA-tubes. DNA was extracted by the standard phenol-chloroform extraction. ApoE genotypes were analysed using polymerase chain reaction (PCR) as described earlier ~5'5~ with slight modifications. In brief, amplification reaction, at the volume of 50pl, consisted of 400ng of genomic DNA, 25 pmol of each primer, 200 pmol/l of each deoxynucleoside triphosphate and 1.5 U ofTaq DNA polymerase (Promega, Madison, WI). Buffer concentration was as recommended by the manufacturer (Promega). To relax secondary DNA structures dimethylsulfoxide was added to a final concentration of 5%. The samples were denatured at 96°C for 15 min before adding of the Taq DNA polymerase. The following cycling reaction conditions were repeated 35 times: denaturing at 96°C for 2 rain, annealing at 60°C for 2.2 rain and extending at 73°C for 2.5 min. The reaction was

67

finished with an extra primer extension step at 73°C for 10 min.

Identification of apolipoprotein E genotypes through Hhaldigestion. Eighteen microliters of the PCR-products were digested with 8 U of HhaI (New England Biolabs, Beverly, MA) at 37°C for at least 3 h. Digested DNA fragments were analysed through a 0.5 mm 10% non-denaturing polyacrylamide gel containing 5% glycerol. Electrophoresis was performed at 400 V for 120 min in a Protean II apparatus (Bio-Rad, Richmond, CA). Separated DNA fragments were visualized through ethidium bromide staining.

Statistical analysis We used the analysis of variance (ANOVA) for independent samples to detect differences of means over the control and AD subgroups of ApoE 4/4, 3/4 and 3/3 or 3/2. Duncan method was applied in the post hoc analysis. If the data did not meet the assumptions of parametric methods, we used the Kruskall-Wallis analysis of variance over the four study groups and the Mann-Whitney U-test to determine which two groups differed from each other. The Chi-square test was used for testing the difference in the gender and ApoE genotype distribution. The level of significance is P < 0.05. RESULTS

The A D p a t i e n t s o f different A p o E genotypes did not differ from controls in age or sex (Table 1). The controls h a d longer e d u c a t i o n t h a n the A D patients [ A N O V A , F ( 3 , 3 8 ) = 8 . 2 , P <0.0001]. As expected, M M S E scores were higher in controls t h a n in A D patients [ F ( 3 , 3 8 ) = 13.3, P <0.0001]. The A D subgroups did not differ in age, sex, age at onset, education, or scores of Webster, M M S E a n d H a m i l t o n scales. The A p o E genotypes 4/4, 3/4, 3/3, 3/2 were detected in 5, 9, 11, 1 o f the A D patients a n d in 1, 2, 13, n o n e of the controls, respectively (Chi-square, P > 0.05). The allele frequencies for A D patients a n d controls were: E4 0.365 vs 0.125, E3 0.615 vs 0.875, a n d E2 0.020 vs 0. The difference in allele frequencies was not statistically significant.

Volumetric measures Table 2 presents the m e a n volumes of the regions of interest. All statistical analyses were performed using volumes normalized for the brain area. A N O V A over the controls a n d A D subgroups showed significant differences in the volumes of the right a n d left h i p p o c a m p u s ( P < 0.0001), right a m y g d a l a (0.05) as well as the left frontal lobe ( P < 0.05). The post hoc analysis revealed t h a t the controls h a d larger h i p p o c a m p i t h a n A D subgroups. The controls also h a d the largest amygdaloid volumes, but the difference in the volume o f the right a n d left a m y g d a l a was significant only c o m p a r e d with the A D E4/4 s u b g r o u p ( P < 0.05). The volume of the left frontal lobe was significantly smaller in A D patients with E3/4 a n d E3/3 genotypes c o m p a r e d with controls ( P < 0.05). The A D 4/4 subjects displayed the most pron o u n c e d reduction in the h i p p o c a m p a l a n d amygdaloid volumes. They h a d significantly smaller volumes o f the right h i p p o c a m p u s ( - 54% of control)

M. Lehtovirta et al.

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Table 2. Volumes (cubic centimeters) of the hippocampus, amygdala and frontal lobe in Alzheimer patients of different apolipoprotein E genotypes

Controls (N = 16) Right hippocampus Left hippocampus Right amygdala Left amygdala Right frontal lobe Left frontal lobe

3.71 _+0.11f 3.35 _ 0.11f 1.69 + 0.06 1.85 + 0.06J; 119.96 _ 19.16 118.45 +_ 19.46§

Alzheimer patients Apolipoprotein E genotype 4/4 3/4 (N = 5) (N = 9) 1.69 __+0.55¢ 1,84 + 0.41 1.07 + 0.49t 1.29 + 0.67 104.72 + 18.35 107.48___19.21

2.41 + 0.50 2.04 _+0.49 1.49 + 0.42 1.43 + 0.43 105.70 +__12.56 98.80 + 8.07

3/3 or 3/2 (N = 12)

ANOVA F

2.53 + 0.56 2.20 +_0.70 1.61 ___0.59 1.59 +_0.48 104.06 __+19.12 92.35 + 17.79

19.7"* 13.0"* 2.9* 2.5 0.97 8.2*

Values expressed as mean + S.D. The statistical analysis was performed using volumes normalized for brain area. ANOVA over the study groups; **P < 0.0001, *P < 0.05. Duncan (P < 0.05): fdiffers from all other groups~ :~differs from AD 4/4, §differs from AD 3/3 or 3/2 group.

and the fight amygdala ( - 3 7 % of control) than all the other study groups (Fig. IA, B).

the frontal lobes (P <0.001) in the whole study population, but the correlation was not significant in the AD group.

Memory and cognitive functions The performance of AD subgroups was significantly impaired compared with controls on memory tests, Boston naming, visuospatial functions and executive functions (Table 3). The AD E4/4 patients had lowest scores on delayed memory tests. The AD E4/4 differed significantly from AD patients with E3/3 in delayed recognition of learned words (Duncan, P < 0.05). In the whole study population the hippocampal volumes correlated with scores on delayed word recognition (r = 0.51, P < 0.001 for the right hippocampus; r =0.45, P <0.001 for the left hippocampus) and delayed visual memory (r=0.63, P < 0.0001 for the fight and left hippocampi); the smaller volume the more impaired performance. In the AD group these correlations were also positive, but they did not reach statistical significance. Of executive functions, the performance on the trailmaking test correlated significantly with volume of

Recent development in molecular genetics in AD research has concretely changed our view of the pathogenesis of AD and further encouraged study of the heterogeneity of this disease. The identification of AD subtypes might have an impact on patients' response to various treatment strategies, as has been implicated by experience from drug trials with cholinergic agents in A D . 3 This study focused on the measurement of volumes of the hippocampus, amygdala, frontal lobes as well as the profile of neuropsychological dysfunctions in AD patients of different ApoE genotypes. In accordance with earlier r e p o r t s , n'19'2°'46 we found that the AD patients had smaller volumes of the hippocampi and also of the left frontal lobe. In this study, we evaluated the contribution of the ApoE genotype to the heterogeneity of AD, and tested the hypothesis that AD

(A) Volume of the right hippocampus (cm 3)

(B) Volume of the right amygdala (cm 3)

DISCUSSION

3

5

2.5

2

i •

!

•

1.5

i

t 1

0.5

AD 4/4

AD 3/4

AD 3/3,3/2

C

AD 4/4

AD 3/4

AD 3/3,3/2

Fig. 1. Volumes of the right hippocampus (cm3), (A) and the right amygdala (cm3); (B) for Alzheimer patients (AD) with different apolipoprotein E genotypes and controls (C). The statistical analysis was performed using volumes normalized for brain area. Controls and AD 4/4 patients differ significantlyfrom all other groups (ANOVA/Duncan, P < 0.05).

C

Hippocampus, apolipoprotein E and Alzheimer's disease

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Table 3. Scores of tests assessing memory and cognitive functions for controls and Alzheimer patients with different apolipoprotein E genotypes

Immediate memory List learning Visual reproduction Delayed memory List learning Visual reproduction Boston naming Visuospatial functions Copy a cube Clock setting Block design Executive functions Trail making A Trail making B Wisconcin card sorting Verbal fluency Praxic functions

Controls (N = 16)

4/4 (N = 5)

Alzheimer patients 3/4 (N = 9) 3/3 (N = 12)

44.6 + 6.2 12.1 + 2.7

17.2+6.1 4.8___0.4

18.3+9.3 3.3+2.1

20.8__+ 12.1 5.4+2.9

25.5** 38.0**

I0.0 + 0.0 11.8 _ 3.6 24.4 + 3.1

3.7 __+3.6t 0.6__+0.9 19.4 _ 0.0

5.2 + 4.0 1.0+ 1.4 13.4 _ 6.9

7.3 + 2.6 2.2+2.8 14.4 __+6.7

10.0** 54.1"* 8.3*

12.6 + 1.3 12.0 + 0.0 33.3 _ 6.5

11.4 + 1.7 9.0__+4.1 16.4 __+3.6

9.0 + 4.4 5.8__+2.6 7.6 + 9.5

9.6 + 2.2 8.6+3.8 14.5 + 8.4

16.5":~ 19.6"~ 22.7**

46.2 __+19.6 196.1 _ 70.8 4.4 + 2.1 55.6 + 18.2

90.4 + 36.9 275.0 + 55.0 1.4_+ 1.8 24.6 + 10.5 37.6 __+5.7

130.6 + 31.8 300.0 + 0.0 0.5+0,8 20.0 _ 3.8 31.6 + 5.6

108.7 + 36.8 266.8 + 60.7 1.0_ 1.2 16.0 + 13.0 35.7 __+6.0

19.2"* 41.4** 21.6"*~ 20.2** NS

r

Results are expressed as mean _ S.D. ANOVA over the study groups: **P < 0.0001, *P < 0.001, Duncan post hoc analysis shows that controls differ from AD subgroups in all test scores. Duncan tAD 4/4 differs from controls and AD 3/3. ++Kruskall-Wallisanalysis of variance over the four study groups followed by Mann Whitney U-test.

patients carrying the ApoE E4 allele might have a more prominent shrinkage of the hippocampus and a more severe memory loss than AD patients without the E4 allele, despite equal global severity of dementia. The major finding of the present study was that the AD E4/4 patients had the most prominent volume loss in the hippocampus and amygdala, and differed significantly from the AD E3/4 and AD E3/3 or E3/2 groups in the volume of the right hippocampus and right amygdala. The AD E4/4 patients also had lowest scores on tests assessing long-term verbal and visual memory. The difference was significant in the recognition of learned words compared with controls and AD patients without the E4 allele. In a previous study, patients with temporal lobe damage have shown more rapid forgetting and worse recognition of learned material than controls. 4~ In accordance with these findings, we detected impaired recognition of learned words in the AD E4/4 patients and volume loss in the hippocampus. Otherwise, the profile of cognitive dysfunctions did not differ between the AD subgroups with different ApoE genotypes. Pathological studies have shown that the cholinergic deficit, plaques and tangles are generally symmetrical, but some AD patients show asymmetries that do not favour either hemisphereY '39 Some regional cerebral blood flow studies have demonstrated predominantly left-sided hypoperfusion in AD patients. 3~ In the interpretation of our results, we want to emphasize the small number of patients, particularly, in the AD E4/4 group. The small sample size and consequently a low statistical power may account for the findings of the statistically significant difference for the right hippocampus but not for the left among AD patients with different ApoE

genotypes. Handedness did not explain this result either. We wanted to select AD patients with different ApoE genotypes at the early stage of the disease and of equal global clinical severity. Furthermore, we need to keep in mind the possibility of type I error in the interpretation of one significant difference, that found in delayed word recognition in AD patients with differing ApoE genotypes, among many neuropsychological variables tested. However, we believe that our results encourage study of the relationship of the ApoE genotype and hippocampal volumes in a larger number of AD patients and controls. Previous studies have indicated the role of medical temporal lobe structures, particularly, of the hippocampus in the processing of certain type of memory. 4~ In the combined group of AD patients and controls, the performance on verbal and visual memory tests was strongly related to the hippocampal volumes. In the AD group, the correlations between the hippocampal volumes and scores on visual and verbal memory tests did not reach statistical significance. This may be partly due to the floor effect of the tests used; the prominent memory impairment already at the stage of the disease results in reduction in variability and concentration of the test scores at the lower end of the spectrum. In an earlier study on subjects with age-associated memory impairment, the volume of the right hippocampus was shown to correlate with performance on a visual memory test/7 In epileptic patients, right hippocampal foci were found to result in impairment in visual memory tests and left hippocampal foci were associated with impairment in verbal memory tests. 26 The E4 allele frequency 0.365 found in AD patients of this study is comparable with previously reported frequencies ranging from 0.3642 to 0.50. 49 The AD

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M. Lehtovirta et al.

group of our study was unselected as to family history of the disease and age of onset. Our controls had E4 frequency of 0.125 that is lower than 0.16 reported in other studies. 42'49 Our controls were selected, cognitively intact and healthy without any major diseases or medication. There is significant heterogeneity in the ApoE polymorphism among populations of different ethnic origin.12 A previous study reported E4 allele frequency of 0.24 in middle aged Finnish subjects. 9 The considerably lower frequency found in the present study may be attributed to higher age of the controls (mean 70 years), a high risk for coronary heart disease and increased mortality in middle subjects with E4 allele, and to a small number and selection of controls. The exact role of ApoE in the pathogenesis of AD is unknown. Besides the high frequency of ApoE E4 among AD patients, the role of ApoE is supported by findings of ApoE within amyloid deposits in AD brain, 29,56 increased ApoE m R N A in astrocytes of AD brain, 7 and high avidity binding of cerebrospinal fluid ApoE to synthetic amyloid-fl-protein and the significantly different binding characteristics of the ApoE4 isoform to fl-peptide than the ApoE3 isof o r m . 49 Interestingly, Schmechel et al., 44 reported that the brains of AD patients homozygous for ApoE E4 contain more vascular amyloid deposits and a higher number and density of amyloid and neuritic plaques than those of E3 homozygous patients. Thus, increased accumulation of amyloid-fl-protein in the brains of AD patients carrying the E4 allele might be an alternative explanation for the prominent atrophy of the hippocampus already at the early stage of ~the disease. However, immunohistochemical studies have indicated presence of ApoE in amyloid deposits not only in AD but also in Down's syndrome and Creutzfeldt-Jacob disease 28 also in cases without ApoE E4 allele. 43 In addition, a recent study suggested that ApoE3 binds to tau protein, stabilizes microtubules and prevents formation of paired helical filaments, another hallmark of AD, whereas ApoE4 does not bind to tau and consequently promotes phosphorylation of tau and hence formation of paired helical filaments. 5° Besides the diverse binding of ApoE isoforms to fl-amyloid and tau protein, experimental studies suggest that ApoE is involved in the synaptogenesis. 33 Poirier et al. found that induction of ApoE gene

expression coincided with reactive synaptogenesis and terminal proliferation in the hippocampus after lesioning the entorhinal cortex in rat. 33 Another study 34 suggested that cholesterol released during terminal breakdown is transported via the ApoE transport system to neurons undergoing reinnervation. Their data suggested that the ApoElipoprotein complexes are taken up by low-density lipoprotein receptors, whose density is increased after lesioning, and being used to recycle the cholesterol/cholesterol esters derived from degraded terminals to sprouting and dendrite reorganization. ApoE phenotypes vary in affinity to low-density lipoprotein receptors. 2~ Given the integral part of cholesterol and other lipoprotein transport in the brain for synaptogenesis, it is possible that AD patients differing in ApoE phenotype also have different degrees of capacity for synaptogenesis. We found most severe volume loss, particularly, in the hippocampus but also in the amygdala of AD patients homozygous for ApoE E4. During memory processing, strong synaptogenesis and reorganization normally take place in the hippocampus.1 Therefore, it is possible that ApoE interferes with synaptogenesis contributing to differences in volume loss in AD patients with differing ApoE phenotypes. CONCLUSIONS

We found that AD patients homozygous for ApoE E4 had smaller volumes of the hippocampus and the amygdala than AD patients heterozygous for E4 and those without the E4 allele. ApoE E4/4 patients also showed lowest scores on delayed memory tests. The AD subgroups did not differ in the global clinical severity of dementia or in the profile of neuropsychological deficits. Previous studies have suggested differences in binding of ApoE isoforms to fl-amyloid and tau protein. Whether the larger volume loss in the hippocampus is related to greater loss of synapses, increased accumulation of fl-amyloid and paired helical filaments in the hippocampus of ApoE E4 homozygous patients, is of interest and worth studying. Acknowledgements--The Medical Research Council of the

Academy of Finland supported this study. The authors thank Mrs Seija Hynynen and Pasi Hakulinen BSc for skillful technical help.

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