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Immune response to Aβ-peptides in peripheral blood from patients with Alzheimer's disease and control subjects
Neuroscience Letters 355 (2004) 226–230 www.elsevier.com/locate/neulet
Immune response to Ab-peptides in peripheral blood from patients with Alzheimer’s disease and control subjects Laurence Barila,*, Ludovic Nicolasa,b, Bernard Croisilec, Pierre Croziera, Catherine Hesslerb, Agne`s Sassolasd, Joseph B. McCormicka,1, Emanuelle Trannoyb a
Epidemiology – Global Medical Affairs, Aventis Pasteur, 2 Avenue Pont Pasteur, 69007 Lyon, France Immunology – Research, Aventis Pasteur, 1541 avenue Marcel me´rieux, 69280 Marcy l’Etoile, France c Laboratory of Neuropsychology, Hoˆpital Neurologique et Neurochirurgical Pierre Wertheimer, 59 Boulevard Pinel, 69003 Lyon, France d Laboratory of Biochemistry, Hoˆpital Neurologique et Neurochirurgical Pierre Wertheimer, 59 Boulevard Pinel, 69003 Lyon, France b
Received 28 August 2003; received in revised form 27 October 2003; accepted 27 October 2003
Abstract To investigate the immune response to amyloid b-peptide (Ab: Ab40 and Ab42) in peripheral human blood, sera were obtained from 36 patients with Alzheimer’s disease (AD) and 34 age-matched controls. ELISA assays were used to measure antibody concentrations to Abpeptides. T cell response was assessed using a lymphoproliferation assay. Both AD and control subjects had low and variable concentrations of antibodies against Ab (predominantly IgG1). The mean antibody to Ab concentrations did not differ between groups. No specific T cell response to Ab-peptides was detected. Natural levels of antibodies to Ab in peripheral blood are present in all human subjects and are unlikely to be useful in the identification of patients who would respond to potential AD immune therapy. Specific cellular immune responses to Ab in human blood were not detected. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Immune response; Amyloid b-peptides; Case-control study
Alzheimer’s disease (AD) is characterised by progressive memory loss, confusion, behavioural disorders, language impairments, and spatial disorientation. It is associated with both senile plaques that are formed of an abnormal accumulation of amyloid b-peptide (Ab) and neurofibrillary tangle, and intraneuronal accumulation of paired helical filaments (PHF) from hyperphosphorylated protein t [21]. Over the last 10 years, it has become increasingly clear that senile plaque formation is not simply a marker of neural degeneration, but is an integral component of the pathological process of AD. Studies in patients with early-onset familial AD revealed a number of mutations in the gene encoding the amyloid precursor protein (APP), the precursor of two amyloid b-peptides found in the brain, Ab40 and Ab42. These mutations are all thought to lead to * Corresponding author. Emerging Diseases Epidemiology Unit, Institut Pasteur, 28, rue du Dr Roux, 75015 Paris, France. Tel.: þ33-1-4061-3887; fax: þ 33-1-4568-8876. E-mail address: [email protected] (L. Baril). 1 Present address: University of Texas Houston School of Public Health, Brownsville Regional Campus, TX, USA.
increases in the production of Ab42, the predominant form of amyloid found in the core of senile plaques. Amyloid Ab42 has been shown to possess neurotoxic properties. The demonstrated neurotoxic properties of Ab42 gave rise to the amyloid cascade hypothesis [11], which proposes that Ab42 is the primary trigger for the physiopathological changes observed in patients with AD inducing activation of inflammatory processes, synaptic loss, formation of PHF, and ultimately neuronal death. The immune system is thought to play a significant role in the development of the pathogenic processes underlying AD [23]. Researchers attempting to elucidate this role have, however, found evidence of both protective and deleterious immune effects. Antigen-specific acquired immunity directed against amyloid-b-peptides may play a protective role against the development of AD. Both humoral and cellular immune responses are probably associated with the control of Ab production and its elimination in humans. It has been postulated that these protective mechanisms are deficient in patients with AD [16]. Inflammatory proteins are thought to exacerbate the pathogenic processes by
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.10.071
L. Baril et al. / Neuroscience Letters 355 (2004) 226–230
stimulating the production of Ab, facilitating its aggregation into plaques, and increasing its cytotoxicity [23]. In addition, the search for immune markers in the serum, cerebrospinal fluid (CSF), and brain of patients with AD has revealed a large number of autoantibodies, which led to the hypothesis that AD pathogenesis involves the induction of large-scale autoimmune response [22]. Current treatments for AD provide only modest symptom relief and there is, therefore, a need for the development of therapeutic and prophylactic vaccine strategies that could slow the disease in affected individuals and prevent or delay the development of disease in susceptible individuals. Several investigations have recently been reported using specific immune stimulations in some AD-like mouse models [2,14,19,20]. In particular, studies of the efficacy of immune stimulations have been conducted in transgenic PDAPP mice that exhibit AD-like pathology through the expression of a mutant human APP mini-gene driven by a platelet-derived growth factor promoter. In this model system, immunisation with a human Ab42 peptide (huAb42) prevented the development of amyloid plaques, neuritic dystrophy, and astrogliosis in young PDAPP mice. Immune responses to the huAb42 peptide in older mice also reduced the rate and the extent of these neuropathologies as well as learning and memory impairments. Passive administration of human antibodies to amyloid b-peptide have been shown to enter the central nervous system and adhere to amyloid plaques, resulting in their clearance [2]. Du et al. [6] have identified naturally occurring antibodies directed against Ab in the CSF and the plasma of both patients with AD and healthy controls. Moreover, they reported that Ab antibody concentrations were lower in CSF from patients with AD compared to those seen in healthy control subjects. The lack of clear understanding of the immune responses to Ab and its potential effects have generated concerns that vaccinations of humans with Ab might elicit large-scale T cell activation and antibody production. Before developing Ab42 immunisation as a vaccine intervention for the treatment or prevention of Alzheimer’s disease in humans, it would be instructive to investigate natural immune responses directed against the amyloid peptides Ab40 and Ab42. The characterisation of anti-Ab responses in AD patients and normal subjects was the primary objective of this study. This was a case control study conducted at the Laboratory of Neuropsychology, Pierre Wertheimer Hospital in Lyon (France) between October of 2000 and May of 2001. The study was approved by the hospital ethics committee (Comite´ Consultatif de Protection des Personnes dans la Recherche Biome´dicale Lyon A) before enrolment of any subjects into the study. All subjects gave written informed consent before entering the study. In the case of patients with diagnosed AD, written consent was also simultaneously obtained from a spouse or legal guardian. Sera from young adults were also extracted from Aventis
227
Pasteur sera bank and sera from children were provided by CA Siegrist (CMU, Geneva, Switzerland). These sera from children were obtained for research purposes with full parental consent. All subjects were outpatients, living at home and none received any individual benefit from taking part in the study. The clinical diagnosis of AD in case subjects was done through a series of standardised psychometric tests. In this study, all patients with AD had a history of progressive cognitive impairment, including loss of memory and difficulties in at least one additional cognitive function. Complete blood count, calcium concentration, renal, hepatic and thyroid function tests were normal. Serologies for syphilis and HIV were negative. Computerised tomography brain scans showed diffuse cerebral cortical atrophy or medio-temporal atrophy. None of the patients was hypertensive, none had a history of alcohol abuse or cerebrovascular disease, and Hachinski Ischaemic Scale scores never exceeded four [9]. The patients had a score of less than ten on the Hamilton Depression rating Scale [10]. The neuropsychological battery tests evaluated dementia severity, language, praxis, attention and verbal memory. The severity of illness was assessed by the Mini-Mental-State Examination (MMSE; [8]) and the patients with AD consisted of persons with mild to moderate AD with MMSE ranging from 13 to 22. The overall level of language function was evaluated by a naming task and a verbal fluency task (words beginning with a letter P in 2 min and words in an animal category in 2 min). Limb praxis was evaluated with three symbolic gestures on verbal command and two meaningless movements on visual imitation. Intentional abilities were assessed with the Forward and the Backward Digit spans from the Wechsler Memory Scale. Memory evaluation consisted of: (1) a cued-verbal memory task consisting of the free and cued recall of a five-word list (the five-word test [7]); and (2) a verbal memory task: immediate and delayed recall of a 22information story [1]. Likewise, the diagnosis of ‘probable’ AD was made in accordance with the criteria specified by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Associations (NINCDS & ADRDA) [17]. Control subjects were healthy volunteers with a normal MMSE, matched by age (^ 5 years) to each case subject and with no genetic link with the case subject. Apolipoprotein E (ApoE) phenotypes were determined by isoelectrofocusing in immobilised pH gradients with silver staining [3]. ApoE genotypes were determined by PCR amplification of DNA fragments (exon 4) and RFLP by restriction endonuclease (Hha I) followed by polyacrylamide gel electrophoresis [15]. The concentrations of IgG to Ab42 and Ab40 were assessed by an ELISA assay. Briefly, ELISA plates were coated with a solution of 0.1 mg/ml of either Ab42 or Ab40 (Bachem, Budendorf, Switzerland: references H1368 and H1194, respectively) in coating buffer (carbonate/bicarbo-
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nate, 0.05M, pH 9.6; 100 ml in each well). A range of six dilutions of sera was tested for each serum, and goat antihuman IgG conjugated with horseradish peroxidase (HRP) were used for the detection (KPL, Gaithersburg, MD, USA: reference WH068). Optical density was read at 450 nm. A small number of individuals expressing high IgG responses to Ab42 (n ¼ 12) were selected from the total patient population (seven cases and five control subjects) to measure IgG subclass responses against Ab42 by ELISA (using goat anti-human Ig1, 2, 3 or 4 conjugated with HRP from CLB-TEBU, San Francisco, CA, USA). A lymphoproliferation assay was performed to assess the specific T cell response to a number of different Abpeptides: the whole protein Ab40 (Bachem, 2 mg/ml for the protein), and a pool of seven overlapping peptides of 15 amino acids in length taken from Ab40 (Neosystem, Strasbourg, France: 2 mg/ml for each peptide). Tetanus toxoid (TT: 10 mg/ml), which induces specific T cell activation, and staphylococcal enterotoxin B (SEB: 2 mg/ml), which non-specifically activates T cell, were used as positive controls. The induction of lymphocyte proliferation was assessed by measuring the 3H-thymidine incorporation. All assays were run in triplicate. A response was considered as positive when the proliferation index (counts per minute (CPM) in the tested peptide/CPM in the media control) was superior to 3. In the absence of a standard (i.e. a hyperimmune serum sample of known titer), the antibody concentrations were expressed using arbitrary values: optical density at the 1/100 dilution calculated from the linear regression and multiplied by 1000. The linear regression integrated all points of dilution for which the squared correlation coefficient (rb) was superior to 0.9. After log10 transformation, the mean concentrations of the studied antibodies for each group were calculated. Data sets were analysed with STATA software (Release 7, StataCorp., College Station, TX, USA). Baseline demographic data of the 36 patients with AD and 34 age-matched control subjects enrolled into the study are summarised in Table 1. No statistical difference could be shown between the mean ages of the two subject groups. As would be expected, the MMSE was found to differ significantly between the two subject groups (Kruskal – Wallis test for the equality of medians, P , 0:01). Table 1 also summarised the distribution of ApoE phenotype among the subjects. ApoE E4 was found in 61% of patients with AD compared to 15% of control subjects (2-sided Fisher’s exact test, P ¼ 0:0002). The most frequent phenotype was E3/E4 in patients with AD and E3/E3 in control subjects. The sera from both patients with AD and control subjects contained low and variable levels of antibodies to Ab42 and Ab40. The data are summarised in Table 2 and in Fig. 1. Comparison of IgG concentrations to Ab42 and Ab40 did not show any significant difference between the two subject groups (Student’s t-tests between case and control groups for Ab42 and Ab40: P ¼ 0:056 and P ¼ 0:19, respectively). Comparisons of the log-transformed variances showed that
Table 1 Baseline demographic data and repartition of Apolipoprotein E (ApoE) isoforms
Age (years) Mean Range Sex Male/female MMS score Median 25–75% interquartiles Apo E phenotype E2/E2 E2/E3 E3/E3 E4/E4 E2/E4 E3/E4 E4 phenotype Yes (%) No (%)
Cases (n ¼ 36)
Controls (n ¼ 34)
76.5 66 –86
72.0 63 –82
13/23
7/27
17 13 –22
29 28 –30
0 3 11 5 1 16
1 3 24 1 0 4
22 (61.1) 14 (38.9)
5 (15.1) 28 (84.9)
the variances of IgG to both Ab42 and Ab40 were higher in the patients with AD than in the control group (F test for the homogeneity of variances, P , 1024 and P ¼ 0:016, respectively). These differences in variance were primarily due to the small number of patients with AD with of very high anti-Ab concentrations. As shown in Fig. 1, a total of eight patients with AD had antibody to Ab42 superior to the highest concentration (IgG titer ¼ 1400) in the control group and a total of four patients with AD had antibody concentrations to Ab40 superior to the highest concentration (IgG titer ¼ 1600) in the control group. Those patients did not differ clinically from the other patients with AD. These data suggest that human peripheral blood contains antibodies to Ab of various affinities that bind weakly to Ab peptides. The distribution of the different IgG subclasses (IgG1, IgG2, IgG3, and IgG4) in sera was assessed in a subset of 12 individuals expressing high IgG responses to Ab42. IgG1 was the predominate IgG subclass in both patients with AD and control subjects, IgG4 responses were very low in both Table 2 Log10 IgG mean concentrations (MC) to Ab40 and Ab42 in patients with AD and control subjects Cases (n ¼ 36)
Controls (n ¼ 34)
Log10 IgG to Ab42 MC (^SD)a Range
2.95 (^0.39) 2.34–4.08
2.83 (^0.19) 2.38–3.22
Log10 IgG to Ab40 MC (^SD) Range
2.83 (^0.29) 2.30–3.59
2.78 (^0.19) 2.40–3.20
a
SD, standard deviation.
L. Baril et al. / Neuroscience Letters 355 (2004) 226–230
Fig. 1. Distribution of IgG titres to Ab42 and to Ab40 (ordinates) in cases and control subjects (abscissas).
groups. In order to investigate whether there was a relationship between age and the concentration of IgG to Ab42, sera from 15 children (age range: 1 –10 years), 26 young adults (age range: 20 –48 years), and a randomised sample of 20 elderly patients from the case-control study (ten case and ten control subjects) were analysed. These data are summarised in Table 3. Low levels of IgG directed against Ab42 were found in the three age-classes, and no statistical difference in the IgG concentrations was observed between any two of the three groups. T cell proliferative responses to Ab40 and to a pool of seven overlapping amyloid peptide fragments were investigated. Proliferative responses of peripheral blood T cells from AD patients and from control subjects were measurable only in response to the two positive control antigens TT and SEB. Proliferative responses to the control antigens suggest that cells both from the AD patients and from the control subjects were viable and functional. It is possible that the lymphoproliferation assay failed to detect an Abspecific T cell response to either Ab40 or the pool of overlapping Ab-peptides as a result of an inadequate sensitivity of the assay due to Ab-specific T cell frequency below the threshold of detection. In addition, in an ELISpot
229
assay, Ab40 failed to stimulate IL-5 or interferon-g cytokines responses in peripheral blood T cells from AD patients or control subjects. In this study, we detected the presence of naturally occurring antibodies to Ab in the sera of patients with AD, age-matched controls, and in healthy children and young adults. No statistical differences in the concentrations of antibodies to Ab could be demonstrated between patients with AD and several control groups. This confirms that no particular population without antibodies to Ab could be used as reference. The results of the present study are consistent with the findings of a larger epidemiological study by Hyman et al. [13] that showed no correlation between antibody concentrations to Ab and the development of AD. The pre-existing antibodies to Ab (even in a young population) are probably not targeted against the senile plaques, but against Ab produced peripherally, such as that produced by platelets [5]. It is becoming increasingly clear that the involvement of the immune system in the pathological processes underlying AD is complex and can be counteractive. Some beneficial effects in mice have been reported from induced antibodies selectively directed against residues of 4– 10 Ab42 that probably target only a small subset of toxic Ab species [18]. This led to the suggestion that the induction of an Abspecific immune response in humans might reduce the deposition of Ab plaques in the human brain. However, a recent Phase II clinical trial of an aggregated Ab42 vaccine was suspended due to the occurrence of aseptic meningoencephalitis in some patients [4]. Recently, Hock et al. demonstrated from vaccinated patients who participated in this trial that the immune sera and CSF stained both plaque and vascular amyloid but not endogenous APP or its physiologic derivatives [12]. In conclusion, natural levels of antibodies to Ab in peripheral blood are unlikely to be useful in the identification of patients who would respond to potential immune therapy. The involvement of the immune system in the neuropathological processes underlying AD should be better characterised before Ab-specific immunisation could be considered as safe for human investigation.
Table 3 Comparison of log10 IgG mean concentrations to Ab42 in children, young adults, and elderly patients Children (n ¼ 15)
Young adults (n ¼ 26)
Elderly (n ¼ 20)a
Log10 IgG to Ab42 Mean concentration (^SD)b Range
2.75 (^0.3) 2.41–3.28
2.70 (^0.26) 2.24– 3.18
2.86 (^0.35) 2.42–3.50
Age (years) Mean (^SD) Range
4 (^3) 1–10
28 (^6) 20–48
74 (^5) 66–81
a b
Randomised sample of ten cases with AD and ten control subjects. SD, standard deviation.
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L. Baril et al. / Neuroscience Letters 355 (2004) 226–230
Acknowledgements We thank the volunteers of the Association RhoˆneAlzheimer for their continued support. We are also indebted to Franc¸oise Borson-Chazot, Nuclear Medicine, Hoˆpital Neurologique et Neurochirurgical Pierre Wertheimer at Lyon for her help in study organisation (subject blood sample collection); Claire-Anne Siegrist for providing the infant sera; the members of the Immunology and Biochemistry Platforms at Aventis Pasteur, Marcy l’Etoile, France for technical advice and to Laurent Pradier and Thierry Canton (Aventis Pharmaceuticals) for their help in setting up the ELISA assays and for their comments on the manuscript.
References [1] J. Barbizet, D. Truscelli, L’histoire du lion (Conside´rations sur la fabulation) (in French), Semaine Hoˆpitaux 28 (1965) 1688–1694. [2] F. Bard, C. Cannon, R. Barbour, R.L. Burke, D. Games, H. Grajeda, T. Guido, K. Hu, J. Huang, K. Johnson-Wood, K. Khan, D. Kholodenko, M. Lee, I. Lieberburg, R. Motter, M. Nguyen, F. Soriano, N. Vasquez, K. Weiss, B. Welch, P. Seubert, D. Schenk, T. Yednock, Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer’s disease, Nat. Med. 6 (2000) 916–919. [3] R. Cartier, A. Sassolas, Apolipoprotein E phenotyping by isoelectric focusing in immobilized pH gradients and silver staining, Electrophoresis 13 (1992) 252–257. [4] E. Check, Nerve inflammation halts trial for Alzheimer’s drug, Nature 415 (2002) 462. [5] M. Di Luca, F. Colciaghi, L. Pastorino, B. Borroni, A. Padovani, F. Cattabeni, Platelets as a peripheral district where to study pathogenetic mechanisms of Alzheimer disease: the case of amyloid precursor protein, Eur. J. Pharmacol. 405 (2000) 277 –283. [6] Y. Du, R. Dodel, H. Hampel, K. Buerger, S. Lin, B. Eastwood, K. Bales, F. Gao, H.J. Moeller, W. Oertel, M. Farlow, S. Paul, Reduced levels of amyloid beta-peptide antibody in Alzheimer disease, Neurology 57 (2001) 801–805. [7] B. Dubois, J. Touchon, F. Portet, P.J. Ousset, B. Vellas, B. Michel, ‘The five words’: a simple and sensitive test for diagnosis of Alzheimer’s disease (in French), Presse. Med. 31 (2002) 1696–1699. [8] M.F. Folstein, S.E. Folstein, P.R. McHugh, ‘Mini-mental state’. A practical method for grading the cognitive state of patients for the clinician, J. Psychiatr. Res. 12 (1975) 189 –198. [9] V.C. Hachinski, L.D. Iliff, E. Zilhka, G.H. Du Boulay, V.L. McAllister, J. Marshall, R.W. Russell, L. Symon, Cerebral blood flow in dementia, Arch. Neurol. 32 (1975) 632–637. [10] M. Hamilton, A rating scale for depression, J. Neurol. Neurosurg. Psych. 23 (1960) 56–62.
[11] J.A. Hardy, G.A. Higgins, Alzheimer’s disease: the amyloid cascade hypothesis, Science 256 (1992) 184 –185. [12] C. Hock, U. Konietzko, A. Papassotiropoulos, A. Wollmer, J. Streffer, R.C. Von Rotz, G. Davey, E. Moritz, R.M. Nitsch, Generation of antibodies specific for b-amyloid by vaccination of patients with Alzheimer disease, Nat. Med. 8 (2002) 1270–1275. [13] B.T. Hyman, C. Smith, I. Buldyrev, C. Whelan, H. Brown, M.X. Tang, R. Mayeux, Autoantibodies to amyloid-beta and Alzheimer’s disease, Ann. Neurol. 49 (2001) 808–810. [14] C. Janus, J. Pearson, J. McLaurin, P.M. Mathews, Y. Jiang, S.D. Schmidt, M.A. Chishti, P. Horne, D. Heslin, J. French, H.T. Mount, R.A. Nixon, M. Mercken, C. Bergeron, P.E. Fraser, P. St GeorgeHyslop, D. Westaway, A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease, Nature 408 (2000) 979–982. [15] B. Maekawa, T.G. Cole, R.L. Seip, D. Bylund, Apolipoprotein E genotyping methods for the clinical laboratory, J. Clin. Lab. Anal. 9 (1995) 63– 69. [16] F. Marx, I. Blasko, M. Pavelka, B. Grubeck-Loebenstein, The possible role of the immune system in Alzheimer’s disease, Exp. Gerontol. 33 (1998) 871 –881. [17] G. McKhann, D. Drachman, M. Folstein, R. Katzman, D. Price, E.M. Stadlan, Clinical diagnosis of Alzheimer’s disease: report of NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s disease, Neurology 34 (1984) 939–944. [18] J. McLaurin, R. Cecal, M.E. Kierstead, X. Tian, A.L. Phinney, M. Manea, J.E. French, M.H. Lambermon, A.A. Darabie, M.E. Brown, C. Janus, M.A. Chishti, P. Horne, D. Westaway, P.E. Fraser, H.T. Mount, M. Przybylski, P. St George-Hyslop, Therapeutically effective antibodies against amyloid-b peptide target amyloid-b residues 4– 10 and inhibit cytotxicity and fibrillogenesis, Nat. Med. 8 (2002) 1263–1269. [19] D. Morgan, D.M. Diamond, P.E. Gottschall, K.E. Ugen, C. Dickey, J. Hardy, K. Duff, P. Jantzen, G. DiCarlo, D. Wilcock, K. Connor, J. Hatcher, C. Hope, M. Gordon, G.W. Arendask, A beta peptide vaccination prevents memory loss in an animal model of Alzheimer’s disease, Nature 408 (2000) 982–985. [20] D. Schenk, R. Barbour, W. Dunn, G. Gordon, H. Grajeda, T. Guido, K. Hu, J. Huang, K. Johnson-Wood, D. Kholodenko, M. Lee, Z. Liao, I. Lieberburg, R. Motter, L. Mutter, F. Soriano, G. Shopp, N. Vasquez, C. Vandevert, S. Walker, M. Wogulis, T. Yednock, D. Games, P. Seubert, Immunization with amyloid-beta attenuates Alzheimerdisease-like pathology in the PDAPP mouse, Nature 400 (1999) 173 –177. [21] D.J. Selkoe, The genetics and molecular pathology of Alzheimer’s disease: role of amyloid and the presenilins, Neurol. Clin. 18 (2000) 903 –922. [22] V.K. Singh, Neuroautoimmunity: pathogenic implications for Alzheimer’s disease, Gerontology 43 (19997) 79–94. [23] T. Wyss-Coray, L. Mucke, Inflammation in neurodegenerative disease – a double-edge sword, Neuron 35 (2002) 419 –432.
Immune response to Ab-peptides in peripheral blood from patients with Alzheimer’s disease and control subjects Laurence Barila,*, Ludovic Nicolasa,b, Bernard Croisilec, Pierre Croziera, Catherine Hesslerb, Agne`s Sassolasd, Joseph B. McCormicka,1, Emanuelle Trannoyb a
Epidemiology – Global Medical Affairs, Aventis Pasteur, 2 Avenue Pont Pasteur, 69007 Lyon, France Immunology – Research, Aventis Pasteur, 1541 avenue Marcel me´rieux, 69280 Marcy l’Etoile, France c Laboratory of Neuropsychology, Hoˆpital Neurologique et Neurochirurgical Pierre Wertheimer, 59 Boulevard Pinel, 69003 Lyon, France d Laboratory of Biochemistry, Hoˆpital Neurologique et Neurochirurgical Pierre Wertheimer, 59 Boulevard Pinel, 69003 Lyon, France b
Received 28 August 2003; received in revised form 27 October 2003; accepted 27 October 2003
Abstract To investigate the immune response to amyloid b-peptide (Ab: Ab40 and Ab42) in peripheral human blood, sera were obtained from 36 patients with Alzheimer’s disease (AD) and 34 age-matched controls. ELISA assays were used to measure antibody concentrations to Abpeptides. T cell response was assessed using a lymphoproliferation assay. Both AD and control subjects had low and variable concentrations of antibodies against Ab (predominantly IgG1). The mean antibody to Ab concentrations did not differ between groups. No specific T cell response to Ab-peptides was detected. Natural levels of antibodies to Ab in peripheral blood are present in all human subjects and are unlikely to be useful in the identification of patients who would respond to potential AD immune therapy. Specific cellular immune responses to Ab in human blood were not detected. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Immune response; Amyloid b-peptides; Case-control study
Alzheimer’s disease (AD) is characterised by progressive memory loss, confusion, behavioural disorders, language impairments, and spatial disorientation. It is associated with both senile plaques that are formed of an abnormal accumulation of amyloid b-peptide (Ab) and neurofibrillary tangle, and intraneuronal accumulation of paired helical filaments (PHF) from hyperphosphorylated protein t [21]. Over the last 10 years, it has become increasingly clear that senile plaque formation is not simply a marker of neural degeneration, but is an integral component of the pathological process of AD. Studies in patients with early-onset familial AD revealed a number of mutations in the gene encoding the amyloid precursor protein (APP), the precursor of two amyloid b-peptides found in the brain, Ab40 and Ab42. These mutations are all thought to lead to * Corresponding author. Emerging Diseases Epidemiology Unit, Institut Pasteur, 28, rue du Dr Roux, 75015 Paris, France. Tel.: þ33-1-4061-3887; fax: þ 33-1-4568-8876. E-mail address: [email protected] (L. Baril). 1 Present address: University of Texas Houston School of Public Health, Brownsville Regional Campus, TX, USA.
increases in the production of Ab42, the predominant form of amyloid found in the core of senile plaques. Amyloid Ab42 has been shown to possess neurotoxic properties. The demonstrated neurotoxic properties of Ab42 gave rise to the amyloid cascade hypothesis [11], which proposes that Ab42 is the primary trigger for the physiopathological changes observed in patients with AD inducing activation of inflammatory processes, synaptic loss, formation of PHF, and ultimately neuronal death. The immune system is thought to play a significant role in the development of the pathogenic processes underlying AD [23]. Researchers attempting to elucidate this role have, however, found evidence of both protective and deleterious immune effects. Antigen-specific acquired immunity directed against amyloid-b-peptides may play a protective role against the development of AD. Both humoral and cellular immune responses are probably associated with the control of Ab production and its elimination in humans. It has been postulated that these protective mechanisms are deficient in patients with AD [16]. Inflammatory proteins are thought to exacerbate the pathogenic processes by
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.10.071
L. Baril et al. / Neuroscience Letters 355 (2004) 226–230
stimulating the production of Ab, facilitating its aggregation into plaques, and increasing its cytotoxicity [23]. In addition, the search for immune markers in the serum, cerebrospinal fluid (CSF), and brain of patients with AD has revealed a large number of autoantibodies, which led to the hypothesis that AD pathogenesis involves the induction of large-scale autoimmune response [22]. Current treatments for AD provide only modest symptom relief and there is, therefore, a need for the development of therapeutic and prophylactic vaccine strategies that could slow the disease in affected individuals and prevent or delay the development of disease in susceptible individuals. Several investigations have recently been reported using specific immune stimulations in some AD-like mouse models [2,14,19,20]. In particular, studies of the efficacy of immune stimulations have been conducted in transgenic PDAPP mice that exhibit AD-like pathology through the expression of a mutant human APP mini-gene driven by a platelet-derived growth factor promoter. In this model system, immunisation with a human Ab42 peptide (huAb42) prevented the development of amyloid plaques, neuritic dystrophy, and astrogliosis in young PDAPP mice. Immune responses to the huAb42 peptide in older mice also reduced the rate and the extent of these neuropathologies as well as learning and memory impairments. Passive administration of human antibodies to amyloid b-peptide have been shown to enter the central nervous system and adhere to amyloid plaques, resulting in their clearance [2]. Du et al. [6] have identified naturally occurring antibodies directed against Ab in the CSF and the plasma of both patients with AD and healthy controls. Moreover, they reported that Ab antibody concentrations were lower in CSF from patients with AD compared to those seen in healthy control subjects. The lack of clear understanding of the immune responses to Ab and its potential effects have generated concerns that vaccinations of humans with Ab might elicit large-scale T cell activation and antibody production. Before developing Ab42 immunisation as a vaccine intervention for the treatment or prevention of Alzheimer’s disease in humans, it would be instructive to investigate natural immune responses directed against the amyloid peptides Ab40 and Ab42. The characterisation of anti-Ab responses in AD patients and normal subjects was the primary objective of this study. This was a case control study conducted at the Laboratory of Neuropsychology, Pierre Wertheimer Hospital in Lyon (France) between October of 2000 and May of 2001. The study was approved by the hospital ethics committee (Comite´ Consultatif de Protection des Personnes dans la Recherche Biome´dicale Lyon A) before enrolment of any subjects into the study. All subjects gave written informed consent before entering the study. In the case of patients with diagnosed AD, written consent was also simultaneously obtained from a spouse or legal guardian. Sera from young adults were also extracted from Aventis
227
Pasteur sera bank and sera from children were provided by CA Siegrist (CMU, Geneva, Switzerland). These sera from children were obtained for research purposes with full parental consent. All subjects were outpatients, living at home and none received any individual benefit from taking part in the study. The clinical diagnosis of AD in case subjects was done through a series of standardised psychometric tests. In this study, all patients with AD had a history of progressive cognitive impairment, including loss of memory and difficulties in at least one additional cognitive function. Complete blood count, calcium concentration, renal, hepatic and thyroid function tests were normal. Serologies for syphilis and HIV were negative. Computerised tomography brain scans showed diffuse cerebral cortical atrophy or medio-temporal atrophy. None of the patients was hypertensive, none had a history of alcohol abuse or cerebrovascular disease, and Hachinski Ischaemic Scale scores never exceeded four [9]. The patients had a score of less than ten on the Hamilton Depression rating Scale [10]. The neuropsychological battery tests evaluated dementia severity, language, praxis, attention and verbal memory. The severity of illness was assessed by the Mini-Mental-State Examination (MMSE; [8]) and the patients with AD consisted of persons with mild to moderate AD with MMSE ranging from 13 to 22. The overall level of language function was evaluated by a naming task and a verbal fluency task (words beginning with a letter P in 2 min and words in an animal category in 2 min). Limb praxis was evaluated with three symbolic gestures on verbal command and two meaningless movements on visual imitation. Intentional abilities were assessed with the Forward and the Backward Digit spans from the Wechsler Memory Scale. Memory evaluation consisted of: (1) a cued-verbal memory task consisting of the free and cued recall of a five-word list (the five-word test [7]); and (2) a verbal memory task: immediate and delayed recall of a 22information story [1]. Likewise, the diagnosis of ‘probable’ AD was made in accordance with the criteria specified by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Associations (NINCDS & ADRDA) [17]. Control subjects were healthy volunteers with a normal MMSE, matched by age (^ 5 years) to each case subject and with no genetic link with the case subject. Apolipoprotein E (ApoE) phenotypes were determined by isoelectrofocusing in immobilised pH gradients with silver staining [3]. ApoE genotypes were determined by PCR amplification of DNA fragments (exon 4) and RFLP by restriction endonuclease (Hha I) followed by polyacrylamide gel electrophoresis [15]. The concentrations of IgG to Ab42 and Ab40 were assessed by an ELISA assay. Briefly, ELISA plates were coated with a solution of 0.1 mg/ml of either Ab42 or Ab40 (Bachem, Budendorf, Switzerland: references H1368 and H1194, respectively) in coating buffer (carbonate/bicarbo-
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nate, 0.05M, pH 9.6; 100 ml in each well). A range of six dilutions of sera was tested for each serum, and goat antihuman IgG conjugated with horseradish peroxidase (HRP) were used for the detection (KPL, Gaithersburg, MD, USA: reference WH068). Optical density was read at 450 nm. A small number of individuals expressing high IgG responses to Ab42 (n ¼ 12) were selected from the total patient population (seven cases and five control subjects) to measure IgG subclass responses against Ab42 by ELISA (using goat anti-human Ig1, 2, 3 or 4 conjugated with HRP from CLB-TEBU, San Francisco, CA, USA). A lymphoproliferation assay was performed to assess the specific T cell response to a number of different Abpeptides: the whole protein Ab40 (Bachem, 2 mg/ml for the protein), and a pool of seven overlapping peptides of 15 amino acids in length taken from Ab40 (Neosystem, Strasbourg, France: 2 mg/ml for each peptide). Tetanus toxoid (TT: 10 mg/ml), which induces specific T cell activation, and staphylococcal enterotoxin B (SEB: 2 mg/ml), which non-specifically activates T cell, were used as positive controls. The induction of lymphocyte proliferation was assessed by measuring the 3H-thymidine incorporation. All assays were run in triplicate. A response was considered as positive when the proliferation index (counts per minute (CPM) in the tested peptide/CPM in the media control) was superior to 3. In the absence of a standard (i.e. a hyperimmune serum sample of known titer), the antibody concentrations were expressed using arbitrary values: optical density at the 1/100 dilution calculated from the linear regression and multiplied by 1000. The linear regression integrated all points of dilution for which the squared correlation coefficient (rb) was superior to 0.9. After log10 transformation, the mean concentrations of the studied antibodies for each group were calculated. Data sets were analysed with STATA software (Release 7, StataCorp., College Station, TX, USA). Baseline demographic data of the 36 patients with AD and 34 age-matched control subjects enrolled into the study are summarised in Table 1. No statistical difference could be shown between the mean ages of the two subject groups. As would be expected, the MMSE was found to differ significantly between the two subject groups (Kruskal – Wallis test for the equality of medians, P , 0:01). Table 1 also summarised the distribution of ApoE phenotype among the subjects. ApoE E4 was found in 61% of patients with AD compared to 15% of control subjects (2-sided Fisher’s exact test, P ¼ 0:0002). The most frequent phenotype was E3/E4 in patients with AD and E3/E3 in control subjects. The sera from both patients with AD and control subjects contained low and variable levels of antibodies to Ab42 and Ab40. The data are summarised in Table 2 and in Fig. 1. Comparison of IgG concentrations to Ab42 and Ab40 did not show any significant difference between the two subject groups (Student’s t-tests between case and control groups for Ab42 and Ab40: P ¼ 0:056 and P ¼ 0:19, respectively). Comparisons of the log-transformed variances showed that
Table 1 Baseline demographic data and repartition of Apolipoprotein E (ApoE) isoforms
Age (years) Mean Range Sex Male/female MMS score Median 25–75% interquartiles Apo E phenotype E2/E2 E2/E3 E3/E3 E4/E4 E2/E4 E3/E4 E4 phenotype Yes (%) No (%)
Cases (n ¼ 36)
Controls (n ¼ 34)
76.5 66 –86
72.0 63 –82
13/23
7/27
17 13 –22
29 28 –30
0 3 11 5 1 16
1 3 24 1 0 4
22 (61.1) 14 (38.9)
5 (15.1) 28 (84.9)
the variances of IgG to both Ab42 and Ab40 were higher in the patients with AD than in the control group (F test for the homogeneity of variances, P , 1024 and P ¼ 0:016, respectively). These differences in variance were primarily due to the small number of patients with AD with of very high anti-Ab concentrations. As shown in Fig. 1, a total of eight patients with AD had antibody to Ab42 superior to the highest concentration (IgG titer ¼ 1400) in the control group and a total of four patients with AD had antibody concentrations to Ab40 superior to the highest concentration (IgG titer ¼ 1600) in the control group. Those patients did not differ clinically from the other patients with AD. These data suggest that human peripheral blood contains antibodies to Ab of various affinities that bind weakly to Ab peptides. The distribution of the different IgG subclasses (IgG1, IgG2, IgG3, and IgG4) in sera was assessed in a subset of 12 individuals expressing high IgG responses to Ab42. IgG1 was the predominate IgG subclass in both patients with AD and control subjects, IgG4 responses were very low in both Table 2 Log10 IgG mean concentrations (MC) to Ab40 and Ab42 in patients with AD and control subjects Cases (n ¼ 36)
Controls (n ¼ 34)
Log10 IgG to Ab42 MC (^SD)a Range
2.95 (^0.39) 2.34–4.08
2.83 (^0.19) 2.38–3.22
Log10 IgG to Ab40 MC (^SD) Range
2.83 (^0.29) 2.30–3.59
2.78 (^0.19) 2.40–3.20
a
SD, standard deviation.
L. Baril et al. / Neuroscience Letters 355 (2004) 226–230
Fig. 1. Distribution of IgG titres to Ab42 and to Ab40 (ordinates) in cases and control subjects (abscissas).
groups. In order to investigate whether there was a relationship between age and the concentration of IgG to Ab42, sera from 15 children (age range: 1 –10 years), 26 young adults (age range: 20 –48 years), and a randomised sample of 20 elderly patients from the case-control study (ten case and ten control subjects) were analysed. These data are summarised in Table 3. Low levels of IgG directed against Ab42 were found in the three age-classes, and no statistical difference in the IgG concentrations was observed between any two of the three groups. T cell proliferative responses to Ab40 and to a pool of seven overlapping amyloid peptide fragments were investigated. Proliferative responses of peripheral blood T cells from AD patients and from control subjects were measurable only in response to the two positive control antigens TT and SEB. Proliferative responses to the control antigens suggest that cells both from the AD patients and from the control subjects were viable and functional. It is possible that the lymphoproliferation assay failed to detect an Abspecific T cell response to either Ab40 or the pool of overlapping Ab-peptides as a result of an inadequate sensitivity of the assay due to Ab-specific T cell frequency below the threshold of detection. In addition, in an ELISpot
229
assay, Ab40 failed to stimulate IL-5 or interferon-g cytokines responses in peripheral blood T cells from AD patients or control subjects. In this study, we detected the presence of naturally occurring antibodies to Ab in the sera of patients with AD, age-matched controls, and in healthy children and young adults. No statistical differences in the concentrations of antibodies to Ab could be demonstrated between patients with AD and several control groups. This confirms that no particular population without antibodies to Ab could be used as reference. The results of the present study are consistent with the findings of a larger epidemiological study by Hyman et al. [13] that showed no correlation between antibody concentrations to Ab and the development of AD. The pre-existing antibodies to Ab (even in a young population) are probably not targeted against the senile plaques, but against Ab produced peripherally, such as that produced by platelets [5]. It is becoming increasingly clear that the involvement of the immune system in the pathological processes underlying AD is complex and can be counteractive. Some beneficial effects in mice have been reported from induced antibodies selectively directed against residues of 4– 10 Ab42 that probably target only a small subset of toxic Ab species [18]. This led to the suggestion that the induction of an Abspecific immune response in humans might reduce the deposition of Ab plaques in the human brain. However, a recent Phase II clinical trial of an aggregated Ab42 vaccine was suspended due to the occurrence of aseptic meningoencephalitis in some patients [4]. Recently, Hock et al. demonstrated from vaccinated patients who participated in this trial that the immune sera and CSF stained both plaque and vascular amyloid but not endogenous APP or its physiologic derivatives [12]. In conclusion, natural levels of antibodies to Ab in peripheral blood are unlikely to be useful in the identification of patients who would respond to potential immune therapy. The involvement of the immune system in the neuropathological processes underlying AD should be better characterised before Ab-specific immunisation could be considered as safe for human investigation.
Table 3 Comparison of log10 IgG mean concentrations to Ab42 in children, young adults, and elderly patients Children (n ¼ 15)
Young adults (n ¼ 26)
Elderly (n ¼ 20)a
Log10 IgG to Ab42 Mean concentration (^SD)b Range
2.75 (^0.3) 2.41–3.28
2.70 (^0.26) 2.24– 3.18
2.86 (^0.35) 2.42–3.50
Age (years) Mean (^SD) Range
4 (^3) 1–10
28 (^6) 20–48
74 (^5) 66–81
a b
Randomised sample of ten cases with AD and ten control subjects. SD, standard deviation.
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Acknowledgements We thank the volunteers of the Association RhoˆneAlzheimer for their continued support. We are also indebted to Franc¸oise Borson-Chazot, Nuclear Medicine, Hoˆpital Neurologique et Neurochirurgical Pierre Wertheimer at Lyon for her help in study organisation (subject blood sample collection); Claire-Anne Siegrist for providing the infant sera; the members of the Immunology and Biochemistry Platforms at Aventis Pasteur, Marcy l’Etoile, France for technical advice and to Laurent Pradier and Thierry Canton (Aventis Pharmaceuticals) for their help in setting up the ELISA assays and for their comments on the manuscript.
References [1] J. Barbizet, D. Truscelli, L’histoire du lion (Conside´rations sur la fabulation) (in French), Semaine Hoˆpitaux 28 (1965) 1688–1694. [2] F. Bard, C. Cannon, R. Barbour, R.L. Burke, D. Games, H. Grajeda, T. Guido, K. Hu, J. Huang, K. Johnson-Wood, K. Khan, D. Kholodenko, M. Lee, I. Lieberburg, R. Motter, M. Nguyen, F. Soriano, N. Vasquez, K. Weiss, B. Welch, P. Seubert, D. Schenk, T. Yednock, Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer’s disease, Nat. Med. 6 (2000) 916–919. [3] R. Cartier, A. Sassolas, Apolipoprotein E phenotyping by isoelectric focusing in immobilized pH gradients and silver staining, Electrophoresis 13 (1992) 252–257. [4] E. Check, Nerve inflammation halts trial for Alzheimer’s drug, Nature 415 (2002) 462. [5] M. Di Luca, F. Colciaghi, L. Pastorino, B. Borroni, A. Padovani, F. Cattabeni, Platelets as a peripheral district where to study pathogenetic mechanisms of Alzheimer disease: the case of amyloid precursor protein, Eur. J. Pharmacol. 405 (2000) 277 –283. [6] Y. Du, R. Dodel, H. Hampel, K. Buerger, S. Lin, B. Eastwood, K. Bales, F. Gao, H.J. Moeller, W. Oertel, M. Farlow, S. Paul, Reduced levels of amyloid beta-peptide antibody in Alzheimer disease, Neurology 57 (2001) 801–805. [7] B. Dubois, J. Touchon, F. Portet, P.J. Ousset, B. Vellas, B. Michel, ‘The five words’: a simple and sensitive test for diagnosis of Alzheimer’s disease (in French), Presse. Med. 31 (2002) 1696–1699. [8] M.F. Folstein, S.E. Folstein, P.R. McHugh, ‘Mini-mental state’. A practical method for grading the cognitive state of patients for the clinician, J. Psychiatr. Res. 12 (1975) 189 –198. [9] V.C. Hachinski, L.D. Iliff, E. Zilhka, G.H. Du Boulay, V.L. McAllister, J. Marshall, R.W. Russell, L. Symon, Cerebral blood flow in dementia, Arch. Neurol. 32 (1975) 632–637. [10] M. Hamilton, A rating scale for depression, J. Neurol. Neurosurg. Psych. 23 (1960) 56–62.
[11] J.A. Hardy, G.A. Higgins, Alzheimer’s disease: the amyloid cascade hypothesis, Science 256 (1992) 184 –185. [12] C. Hock, U. Konietzko, A. Papassotiropoulos, A. Wollmer, J. Streffer, R.C. Von Rotz, G. Davey, E. Moritz, R.M. Nitsch, Generation of antibodies specific for b-amyloid by vaccination of patients with Alzheimer disease, Nat. Med. 8 (2002) 1270–1275. [13] B.T. Hyman, C. Smith, I. Buldyrev, C. Whelan, H. Brown, M.X. Tang, R. Mayeux, Autoantibodies to amyloid-beta and Alzheimer’s disease, Ann. Neurol. 49 (2001) 808–810. [14] C. Janus, J. Pearson, J. McLaurin, P.M. Mathews, Y. Jiang, S.D. Schmidt, M.A. Chishti, P. Horne, D. Heslin, J. French, H.T. Mount, R.A. Nixon, M. Mercken, C. Bergeron, P.E. Fraser, P. St GeorgeHyslop, D. Westaway, A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease, Nature 408 (2000) 979–982. [15] B. Maekawa, T.G. Cole, R.L. Seip, D. Bylund, Apolipoprotein E genotyping methods for the clinical laboratory, J. Clin. Lab. Anal. 9 (1995) 63– 69. [16] F. Marx, I. Blasko, M. Pavelka, B. Grubeck-Loebenstein, The possible role of the immune system in Alzheimer’s disease, Exp. Gerontol. 33 (1998) 871 –881. [17] G. McKhann, D. Drachman, M. Folstein, R. Katzman, D. Price, E.M. Stadlan, Clinical diagnosis of Alzheimer’s disease: report of NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s disease, Neurology 34 (1984) 939–944. [18] J. McLaurin, R. Cecal, M.E. Kierstead, X. Tian, A.L. Phinney, M. Manea, J.E. French, M.H. Lambermon, A.A. Darabie, M.E. Brown, C. Janus, M.A. Chishti, P. Horne, D. Westaway, P.E. Fraser, H.T. Mount, M. Przybylski, P. St George-Hyslop, Therapeutically effective antibodies against amyloid-b peptide target amyloid-b residues 4– 10 and inhibit cytotxicity and fibrillogenesis, Nat. Med. 8 (2002) 1263–1269. [19] D. Morgan, D.M. Diamond, P.E. Gottschall, K.E. Ugen, C. Dickey, J. Hardy, K. Duff, P. Jantzen, G. DiCarlo, D. Wilcock, K. Connor, J. Hatcher, C. Hope, M. Gordon, G.W. Arendask, A beta peptide vaccination prevents memory loss in an animal model of Alzheimer’s disease, Nature 408 (2000) 982–985. [20] D. Schenk, R. Barbour, W. Dunn, G. Gordon, H. Grajeda, T. Guido, K. Hu, J. Huang, K. Johnson-Wood, D. Kholodenko, M. Lee, Z. Liao, I. Lieberburg, R. Motter, L. Mutter, F. Soriano, G. Shopp, N. Vasquez, C. Vandevert, S. Walker, M. Wogulis, T. Yednock, D. Games, P. Seubert, Immunization with amyloid-beta attenuates Alzheimerdisease-like pathology in the PDAPP mouse, Nature 400 (1999) 173 –177. [21] D.J. Selkoe, The genetics and molecular pathology of Alzheimer’s disease: role of amyloid and the presenilins, Neurol. Clin. 18 (2000) 903 –922. [22] V.K. Singh, Neuroautoimmunity: pathogenic implications for Alzheimer’s disease, Gerontology 43 (19997) 79–94. [23] T. Wyss-Coray, L. Mucke, Inflammation in neurodegenerative disease – a double-edge sword, Neuron 35 (2002) 419 –432.