Cerebrospinal fluid apolipoprotein E (apoE) levels in Alzheimer's disease patients are increased at follow up and show a correlation with levels of tau protein

Neuroscience Letters 229 (1997) 85–88

Cerebrospinal fluid apolipoprotein E (apoE) levels in Alzheimer’s disease patients are increased at follow up and show a correlation with levels of tau protein Maria Lindh a, Mari Blomberg b, Malene Jensen a, Hans Basun b, Lars Lannfelt a, Benita Engvall a, Hubert Scharnagel c, Winfried Ma¨rz c, Lars-Olof Wahlund b, Richard F. Cowburn a ,* a

Karolinska Institute, Department of Clinical Neurosciences and Family Medicine, Section for Geriatric Medicine, Novum, KFC, plan 4, S-141 86 Huddinge, Sweden b Karolinska Institute, Department of Clinical Neurosciences and Family Medicine, Section for Geriatric Medicine, Huddinge University Hospital, S-141 86 Huddinge, Sweden c Klinikum der Albert Ludwig Universita¨t, Medizinische Universita¨tsklinik, Abteilung Klinische Chemie-Zentrallabor, Hugstetterstrasse 55, D-79106 Freiburg, Germany Received 12 May 1997; received in revised form 30 May 1997; accepted 30 May 1997

Abstract Apolipoprotein E (apoE) levels were compared in cerebrospinal fluid (CSF) taken on two occasions, with an average 15 months follow up, from groups of patients with Alzheimer’s disease (AD; n = 18), mild cognitive impairment (MCI; n = 9) and other dementia disorders (ODD; n = 9). In these groups, CSF apoE levels were between 2–3-fold higher than values for a group of 27 healthy age-matched controls. CSF apoE levels in the AD group were significantly increased at follow up, compared to levels obtained on the first sampling occasion. For the same cases it had been shown previously that CSF tau protein levels were increased at follow up [Blomberg, M., Jensen, M., Basun, H., Lannfelt, L. and Wahlund, L-O., Neurosci. Lett., 214 (1996) 163–166]. The AD, but not MCI, ODD or control groups, also showed statistically significant correlations between CSF apoE and tau protein levels at both the first (r = 0.585, P , 0.01) and follow up (r = 0.695, P . 0.001) samplings. It is concluded that CSF measures of both apoE and tau may reflect an intimate relationship between these two proteins in AD and could prove useful in monitoring the progression of this condition.  1997 Elsevier Science Ireland Ltd. Keywords: Alzheimer’s disease; Apolipoprotein E; Cerebrospinal fluid; Diagnostic marker; Tau protein

Apolipoprotein E (apoE) is the major apolipoprotein in the central nervous system where it is involved in the mobilisation and redistribution of cholesterol necessary for the maintenance of myelin and neuronal membranes during development and following injury [5]. ApoE exists as three major isoforms encoded by the e2, e3 and e4 alleles. A wealth of genetic and epidemiological evidence indicates that inheritance of the e4 allele provides a major risk factor for the development of Alzheimer’s disease (AD) in numerous populations world-wide (see Ref. [12] for review). Recently, apoE levels were shown to be increased in cerebrospinal fluid (CSF) from patients with late-onset AD, compared with control cases without any sign of neu* Corresponding author. Tel.: +46 8 58583884; fax: +46 8 58583880; e-mail: [email protected]

ropsychiatric disease [10]. This, together with reports that apoE levels are increased also in CSF from patients with other neurological and psychiatric disorders [3,10] suggests that CSF apoE measures may provide a general reflection of neuronal damage and inflammatory reactions in the brain [10]. In the present study, we re-assessed the usefulness of CSF apoE measures as a diagnostic marker for AD by comparing levels in three patient groups, plus a population of healthy age-matched controls. Given the progressive nature of AD and other neurodegenerative disorders, we designed the study as a follow up, with apoE levels being measured in samples that were taken on two occasions with an average 15 months in between. The patient groups used in the study were those in which it had been shown previously that there was an AD related increase in CSF tau protein levels at

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Table 1 Demographic and clinical data for subjects used Number and gender of subjects

Number of cases according to genotype

e2/3 Alzheimer’s disease Mild cognitive impairment Other dementia disorders Controls

Age at 1st LP (years)

Duration of Follow up symptoms time at 1st LP (months) (months)

MMSE at 1st LP

MMSE at 2nd LP

CDR sum of boxes at 1st LP

CDR sum of boxes at 2nd LP

68.4 ± 10.5 (41–82) 68.1 ± 3.1 (51–78)

30 ± 20 (6–84) 40 ± 48 (1–150)

14 ± 7.5 (1–32) 15 ± 6.9 (1–24)

21 ± 5 (10–27) 27 ± 3 (22–30)

18 ± 6 (6–27)* 26 ± 2 (22–28)

5 (1.5–14) 2 (0.5–5)

7.5 (3–15)*** 3 (0.5–6.5)*

71.3 ± 2.3 (60–81) 66.9 ± 9.5 (45–80)

48 ± 37 (12–120) –

17 ± 5.9 (12–29) –

21 ± 4 (14–27) 29 ± 1 (26–30)

18 ± 7 (8–25) –

5 (1–11)

7 (2–15)**

–

–

e3/3 e3/4 e4/4

18 (16 F/2 M) 9 (6 F/3 M)

0

2

11

5

1

4

3

1

9 (6 F/3 M) 27 (17 F/9 M)

0

5

2

1

3

20

4

0

Values for age, duration, follow up time, MMSE and CDR sum of boxes represent the mean ± SD, with ranges given in parentheses. Note: one case from the other dementia disorders group was not apoE genotyped owing to a lack of material. Significance levels are shown for the comparison of variables at 2nd versus 1st LP; *P , 0.05, **P , 0.01, ***P , 0.001 (paired t-test).

follow up. Therefore, data was also analysed to determine potential relationships between CSF apoE and tau protein levels. CSF samples from 63 individuals were used in the study. These included, 18 cases with AD, nine cases with mild cognitive impairment (MCI), nine with other dementia disorders (ODD; including one case of frontal lobe dementia, four cases of vascular dementia, two patients with progressive primary aphasia with dementia, one subject with Parkinson’s disease with dementia and one case of unspecified dementia) and 27 age-matched healthy controls (see Table 1 for demographic and clinical data of the cases used). Clinical diagnoses were made as described previously [2]. In brief, the diagnosis of AD was made according to DSM IV and NINCDS-ADRDA criteria. NINDS-AIREN and ICD-10 criteria were used for the diagnosis of vascular and frontal lobe dementias, respectively. Those patients having cognitive impairment as assessed by neuropsychological testing but with symptoms not severe enough to fulfil DSM IV criteria were classified as having MCI. Global cognitive functions were assessed using the mini-mental status examination (MMSE). The clinical stage of dementia and the patient’s ability to function in daily life were assessed using a modified form of the clinical dementia rating (CDR) scale (for details see Ref. [2]). Samples of CSF were collected on both occasions by lumbar puncture (LP) in the L3/L4 or L4/L5 interspace. Samples were collected before 1200 h with the patient sitting in an upright position. Samples were centrifuged at 1000 rev./min for 10 min and frozen at −70°C in aliquots of 1 ml until assayed. ApoE levels in CSF were determined using a sandwich enzyme linked immunosorbent assay (ELISA). Microwell plates (Nunc) were coated overnight with primary antibodies (polyclonal anti-human apoE that was affinity purified from goat antiserum (Greiner; Frickenhausen, Germany) using human high density lipoproteins coupled to cyanogen

bromide activated sepharose (Pharmacia)) in a coating buffer (66.7 mM Na2HPO4, 66.7 mM KH2PO4, pH 6.0) at a dilution of 1:1000 (v/v) and a volume of 200 ml/well. Non-specific binding was blocked with 2% bovine serum albumin (BSA; Sigma, order no. 4506) in Dist H2O for 2 h and plates then washed four times with 2% BSA. Standard curves were constructed using duplicate samples of six dilutions of an apoE standard serum. CSF samples were diluted in 7% BSA to be in the linear range of the standard curve and triplicate samples then incubated in the microwell plates for 2 h. Plates were washed four times and 200 ml of secondary antibody (biotinylated primary antibody at a dilution of 1:1500 in 1% BSA) added. After a 1 h incubation, the plates were washed and 200 ml of conjugate (POD, streptavidin conjugated horse radish peroxidase, GIBCO, diluted 1:1500 in 1% BSA) added. Plates were incubated for 1 h and then washed four times. Detection was performed by adding 200 ml substrate solution (ABTS, Boerhinger) and incubating plates for 45 min. Colour reactions were detected in an ELISA reader (Emax, Molecular Devices) at 405 nm. Interand intra-assay coefficients of variation were 12.8 and Table 2 CSF apoE measures at the 1st and 2nd lumbar puncture (LP)

Alzheimer’s disease Mild cognitive impairment Other dementia disorders Controls

CSF [ApoE] at 1st LP (mg/l)

CSF [ApoE] at 2nd LP (mg/l)

4.66 ± 1.96*** 3.86 ± 2.08*

5.45 ± 2.69 *** 4.92 ± 2.19**

5.76 ± 2.93***

5.38 ± 3.15***

1.92 ± 1.49

–

Significance levels are shown for comparison with values for the control group using ANOVA with Fisher’s PLSD. A statistically significant difference (P , 0.05) was also seen for CSF apoE levels between the mild cognitive impairment and other dementia disorders groups at 1st LP: *P , 0.05, **P ≤ 0.01, ***P , 0.001.

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Fig. 1. Comparison of CSF apoE levels at the 1st and 2nd LP in the Alzheimer’s disease, mild cognitive impairment and other dementia disorders groups. Filled circles with continuous lines represent those cases showing increased levels at the 2nd versus 1st LP. Open circles with dashed lines represent cases showing unchanged or decreased levels at the 2nd versus 1st LP. Significance levels are shown for the comparison of levels at the 1st and 2nd LP using paired t-test (**P , 0.01).

11.0%, respectively. Western immunoblotting with recombinant apoE (produced in a baculovirus expression system; PanVera, Madison, WI, USA) confirmed that the goat polyclonal anti human apoE antibody reacted equally against the three different apoE isoforms (data not shown). CSF tau protein determinations and apoE genotypings were performed as described earlier [2] using the Innotest hTau Antigen (Innogenetics, Zwijndrecht, Belgium) and Affigene (Sangtec, Bromma, Sweden) commercial kits, respectively. Intra-assay variability for the tau protein ELISA was below 11.0% [6]. Table 2 shows the mean values (±SD) for apoE levels in CSF taken at both sampling occasions from the AD, MCI and ODD cases, as well as from the population of healthy control cases. For both the 1st and 2nd LP samplings, it was found that CSF apoE levels were statistically significantly higher for the AD, MCI and ODD cases, compared with those for the control subjects. Values for the 1st LP for the AD, MCI and ODD groups were approximately 2.4, 2.0 and 3.0 higher than those for the control cases, respectively. Table 2 also shows that for the AD group, CSF apoE levels were significantly increased (P , 0.01, paired ttest) at the 2nd LP sampling, compared with levels obtained at the 1st LP sampling. The individual values for these cases are presented in Fig. 1, from which it can be seen that 14 of the 18 AD cases showed increased CSF apoE levels at follow up, whereas the remainder showed decreased levels. CSF apoE levels also appeared to be increased in the MCI group at follow up (Table 1; Fig. 1). However, this difference did not reach statistical significance (P = 0.08, paired t-test), with six of the nine MCI cases showing an increase (Fig. 1). CSF apoE levels for the ODD cases were not significantly different between the 1st and 2nd LP samplings (Table 1; Fig. 1). Of the AD cases used in the study, it has previously been shown that 12 individuals with either the e3/e4 or e4/e4 genotype showed increased levels of CSF tau protein at the 2nd compared with the 1st LP [2]. Analysis of the current data according to apoE genotype revealed that of the

AD cases that showed increased CSF apoE levels at follow up, two had an e3/e3 genotype, 10 had an e3/e4 genotype and two cases an e4/e4 genotype. For the MCI cases, four had an e3/e3 genotype, one an unknown genotype and the other two e3/e4 and e4/e4. The three ODD cases that showed increased CSF apoE levels at follow up had e2/e3, e3/e3 and e3/e4 genotypes. It was also found that for the AD group, CSF apoE and tau protein levels showed a strong correlation with each other at both the 1st and 2nd LP sampling occasions (Fig. 2). This correlation appeared stronger at the 2nd (r = 0.695, P , 0.001; simple regression analysis with Fisher’s r to z) than at the 1st (r = 0.585, P , 0.01) LP, due to an increase in both CSF apoE and tau protein [2] levels at follow up. The MCI group showed a trend towards a significant correlation between CSF apoE and tau protein levels at the 1st, but not the 2nd LP (see legend to Fig. 2). No significant correlations were found between the levels of these two proteins in either the ODD or control groups (see legend to Fig. 2). Spearman’s rank analyses of the data did not reveal any

Fig. 2. Correlations between CSF apoE and tau protein levels at 1st and 2nd LP for the Alzheimer’s disease group. Lines of best fit are shown for simple regression analysis of the data. Coefficients (r values) were determined from simple correlation analyses of the data with significances determined by Fisher’s r- to z-tests. No statistically significant correlations were seen between CSF apoE and tau protein levels in either the mild cognitive impairment group (r = 0.592, 0.1 , P , 0.05 at 1st LP; r = 0.187, P>0.1 at 2nd LP), other dementia disorders group (r = −0.310, P>0.1 at 1st LP; r = 0.134, P>0.1 at 2nd LP) or the control group (r = −0.039, P>0.1).

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statistically significant correlations between the 1st or 2nd LP CSF apoE levels and either subject age, duration of symptoms, and MMSE and CDR sum of boxes scores in either the AD, MCI or ODD groups. The present study showing increased apoE CSF levels in AD, ODD and MCI patients, compared to non-disease controls, is in agreement with that of Merched et al. who reported higher apoE levels in CSF from AD cases and individuals with low back pain, motor neuron disease and meningoencephalitis [10]. Together, these data are consistent with the notion that CSF apoE levels may reflect common neurodegenerative processes and inflammatory reactions seen in AD and other disorders [10]. On the other hand, it should be emphasised that other studies have shown decreased [1,7] and unchanged [4,8,13] CSF apoE levels in AD patients. Whatever the reason for the discrepancy between these studies, it is clear that CSF apoE measures alone are unlikely to provide a specific marker for the diagnosis of AD. However, our current and previous data showing increased CSF apoE and tau [2] levels at follow up may indicate that the combined measure of these two proteins could prove useful in monitoring the progression of AD and in the evaluation of therapeutic strategies designed to slow the course of the disease. Further studies will be necessary to determine the disease stage at which increased CSF apoE levels become apparent, as well as the more long term changes in the levels of this protein in AD. It is also of interest that CSF apoE and tau protein levels showed a strong correlation with each other in the AD, but not other patient or control groups. Biochemical studies have shown that apoE binds in an isoform specific manner to tau, with the E3 isoform showing greater avidity [14]. This interaction of apoE3 and tau has been hypothesised to play a role in supporting and stabilising microtubule formation and preventing the hyperphosphorylation of tau [15]. Consistent with this, Nathan et al. have demonstrated that apoE3 enhances and apoE4 inhibits neurite outgrowth in cultured neuroblastoma N-2a cells, the latter being due to microtubule depolymerisation as a possible consequence of less efficient binding to microtubule associated proteins [11]. Such studies point to an intimate relationship between apoE and tau that could be reflected in the concentrations of these two proteins in AD CSF. The observation that CSF apoE levels increase in AD cases at follow up could also be explained by isoform specific binding to tau. In this respect, Nathan et al. also showed that N-2a cells accumulate apoE3 in cell bodies and dendrites to a greater extent than they do apoE4 [11] and more recently, Lovestone et al. demonstrated that COS-7 cells transiently transfected with tau and the low density lipoprotein receptor take up and accumulate apoE3 in the cytoplasm, in preference to apoE4 [9]. This study was funded by the European Union Biomed 2 concerted action programme (contract number bmh4-ct96-0162), the Swedish Medical Research Council (K97-

19X-12244-01A), Stiftelsen fo¨r Gamla Tja¨narinnor, Clas Groschinskys foundation, Loo and Hans Ostermans Foundation and the Swedish Society of Medicine. Anna-Lena Wetterholm and Birgitta Strandberg are thanked for help in collecting samples. [1] Blennow, K., Hesse, C. and Fredman, P., Cerebrospinal fluid apolipoprotein E is reduced in Alzheimer’s disease, NeuroReport, 5 (1994) 2534–2536. [2] Blomberg, M., Jensen, M., Basun, H., Lannfelt, L. and Wahlund, L.O., Increasing cerebrospinal fluid tau levels in a subgroup of Alzheimer patients with apolipoprotein E allele e4 during 14 months follow up, Neurosci. Lett., 214 (1996) 163–166. [3] Carlsson, J., Armstrong, V.W., Reiber, H., Felgenhauer, K. and Seidel, D., Clinical relevance of the quantification of apolipoprotein E in cerebrospinal fluid, Clin. Chim. Acta, 196 (1991) 167–176. ˚ hlin, A. and Nyba¨ck, H., Levels of cere[4] Hahne, S., Nordstedt, C., A brospinal fluid apolipoprotein E in patients with Alzheimer’s disease and healthy control, Neurosci. Lett., 224 (1997) 99–102. [5] Ignatius, M.J., Gebicke-Ha¨rter, P.J., Skene, J.H.P., Schilling, J.W., Weisgraber, K.H., Mahley, R.W. and Shooter, E.M., Expression of apolipoprotein E during nerve degeneration and regeneration, Proc. Natl. Acad. Sci. USA, 83 (1986) 1125–1129. [6] Jensen, M., Basun, H. and Lannfelt, L., Increased cerebrospinal fluid tau in patients with Alzheimer’s disease, Neurosci. Lett., 186 (1995) 189–191. [7] Lande´n, M., Hesse, C., Fredman, P., Regland, B., Wallin, A. and Blennow, K., Apolipoprotein E in cerebrospinal fluid from patients with Alzheimer’s disease and other forms of dementia is reduced without any correlation to the apoE4 isoform, Dementia, 7 (1996) 273–278. [8] Lefranc, D., Vermersch, P., Dallongeville, J., Daems-Monpeurt, C., Petit, H. and Delacourte, A., Relevance of the quantification of apolipoprotein E in the cerebrospinal fluid in Alzheimer’s disease, Neurosci. Lett., 212 (1996) 91–94. [9] Lovestone, S., Anderton, B.H., Hartley, C., Jensen, T.G. and Jo¨rgensen, A.L., The intracellular fate of apolipoprotein E is tau dependent and apoE allele-specific, NeuroReport, 7 (1996) 1005– 1008. [10] Merched, A., Blain, H., Visvikis, S., Herbeth, B., Jeandel, C. and Siest, G., Cerebrospinal fluid apolipoprotein E level is increased in late onset Alzheimer’s disease, J. Neurol. Sci., 145 (1997) 33–39. [11] Nathan, B.P., Chang, K.-C., Bellosta, S., Brisch, E., Ge, N., Mahley, R.W. and Pitas, R.E., The inhibitory effect of apolipoprotein E4 on neurite outgrowth is associated with microtubule depolymerization, J. Biol. Chem., 270 (1995) 19791–19799. [12] Roses, A.D., On the metabolism of apolipoprotein E and the Alzheimer diseases, Exp. Neurol., 132 (1995) 149–156. [13] Rosler, N., Wichart, I. and Jellinger, K.A., Intra vitam lumbar cerebrospinal fluid and serum and ventricular immunoreactive apolipoprotein E in patients with Alzheimer’s disease, J. Neurol. Neurosurg. Psychiatry, 60 (1996) 452–454. [14] Strittmatter, W.J., Saunders, A.M., Goedert, M., Weisgraber, K.H., Dong, L.M., Jakes, R., Huang, D.Y., Pericak-Vance, M., Schmechel, D. and Roses, A.D., Isoform-specific interactions of apolipoprotein E with microtubule associated protein tau: implications for Alzheimer disease, Proc. Natl. Acad. Sci. USA, 91 (1994) 11183–11186. [15] Strittmatter, W.J., Weisgraber, K.H., Goedert, M., Saunders, A.M., Huang, D., Corder, E.H., Dong, L.M., Jakes, R., Alberts, M.J., Gilbert, J.R., Han, S.H., Hulette, C., Einstein, G., Schmechel, D., Pericak-Vance, M.A. and Roses, A.D., Hypothesis: microtubule instability and paired helical filaments formation in the Alzheimer’s disease brain are related to apolipoprotein E genotype, Exp. Neurol., 125 (1994) 163–171.