Blood-borne factors inhibit Alzheimer's β-amyloid fibril formation in vitro

Experimental Neurology 202 (2006) 125 – 132 www.elsevier.com/locate/yexnr

Blood-borne factors inhibit Alzheimer's β-amyloid fibril formation in vitro Kenjiro Ono a , Moeko Noguchi-Shinohara a , Miharu Samuraki a , Yasuko Matsumoto a , Daisuke Yanase a , Kazuo Iwasa a , Hironobu Naiki b,c , Masahito Yamada a,⁎ a b

Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8640, Japan Division of Molecular Pathology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan c CREST of Japan Science and Technology Corporation, Saitama, Japan Received 14 February 2006; revised 28 April 2006; accepted 15 May 2006 Available online 27 June 2006

Abstract Soluble amyloid β-protein (Aβ) does not aggregate to β-amyloid fibrils (fAβ) in the brain of normal humans. We recently found that the cerebrospinal fluid (CSF) from non-Alzheimer's disease (AD) subjects inhibited the formation of fAβ(1–40) and fAβ(1–42) more strongly than that from AD subjects, although the CSF obtained from both groups inhibited the fAβs formation in vitro. Here, we examined the influence of plasma obtained from AD, non-AD and healthy control (CTL) subjects on the formation of fAβ(1–40) and fAβ(1–42) in vitro. Although the plasma obtained from all groups inhibited the formation of fAβ(1–40) and fAβ(1–42), the plasma from non-AD and CTL subjects inhibited the formation of fAβs more strongly than that from AD subjects. These results indicate that the plasma as well as CSF in AD would provide a molecular environment favorable for fAβ formation, suggesting a decrease of specific inhibitory factors and/or increase of specific accelerating factors. © 2006 Elsevier Inc. All rights reserved. Keywords: Alzheimer's disease; Plasma; β-amyloid fibrils; Thioflavin T; Electron microscopy

Introduction Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder manifested by cognitive and memory deterioration, progressive impairment of activities in daily life and a variety of neuropsychiatric symptoms and behavioral disturbances (Cummings, 2004). The disease is pathologically characterized by the abundance of extracellular amyloid plaques, composed primarily of the amyloid β-protein (Aβ), and the intraneuronal accumulation of neurofibrillary tangles, composed of the tau protein (Cummings, 2004). The accumulation of Aβ is considered to cause the progression of AD (Cummings, 2004). Aβ can be detected as a circulating peptide in the plasma and cerebrospinal fluid (CSF) of healthy humans (Mayeux et al., 2003). In AD, it has been postulated that increased production and/or decreased metabolism/clearance of Aβ may be primary events that lead to amyloid plaque deposition and subsequently to the cascade of other neuropathological changes associated with the disease (Du et al., 2003). In ⁎ Corresponding author. Fax: +81 76 234 4253. E-mail address: [email protected] (M. Yamada). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2006.05.019

vitro studies using synthetic Aβ peptide(s) have shown that Aβforming β-amyloid fibrils (fAβ) are considered to be central in the pathogenesis of AD, although neurotoxicity can be demonstrated with pre-fibrillar Aβ aggregates (Harper et al., 1997, 1999; Walsh et al., 1997, 1999) or oligomer (Podlisny et al., 1998). Clearance of soluble Aβ across the blood brain barrier (BBB) is controlled by rapid receptor-mediated transport via advanced glycation end products (Deane et al., 2004) and lowdensity lipoprotein receptor-related protein (Shibata et al., 2000), and by binding of Aβ to transport proteins such as apolipoprotein (apo) E and apoJ, which can influence Aβ sequestration in plasma, brain interstitial fluid and CSF (DeMattos et al., 2004; Fagan et al., 2002). Thus, the equilibrium between Aβ concentrations of plasma and CSF is regulated by these clearance pathways. The mechanisms of fAβ formation in vitro can be explained by the nucleation-dependent polymerization model (Lomakin et al., 1997; Naiki et al., 1997). This model consists of two phases, i.e., nucleation and extension phases. Nucleus formation requires a series of association steps of monomers that are thermodynamically unfavorable, representing the rate-limiting

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step in amyloid fibril formation. Once the nucleus has been formed, further addition of monomers becomes thermodynamically favorable, resulting in the rapid extension of amyloid fibrils. A first-order kinetic in vitro model of fAβ extension has been developed and extension of fAβ has been shown to proceed via the consecutive association of Aβ onto the end of existing fibrils (Lomakin et al., 1997; Naiki and Nakakuki, 1996). Although this model is based on the assumption that Aβ is monomeric in the reaction mixture, the protofibrils (Harper et al., 1997, 1999; Walsh et al., 1997, 1999) would also be consistent with this model. A characteristic sigmoidal timecourse curve of fAβ formation from Aβ at a physiological pH is widely believed to represent the essence of a nucleationdependent polymerization model; that is, the initial lag phase represents the thermodynamically unfavorable nucleus formation and the subsequent growing phase represents the extension reaction (Naiki et al., 1997). We recently found that CSF obtained from non-AD subjects inhibited the formation of fAβ(1–40) and fAβ(1–42) more strongly than that from AD subjects (Ono et al., 2005). In this study, we examined the effect of the plasma obtained from AD, non-AD and healthy control (CTL) subjects on fAβ(1–40) and fAβ(1–42) formation from fresh Aβs at pH 7.5 at 37°C in vitro. Additionally, we examined the degree of fAβ formation after incubation with the plasma obtained from AD subjects (ADplasma) in relation to the degree of fAβ formation after incubation with the CSF, Aβ(1–42) in the CSF, Aβ(1–42), albumin (Alb) and IgG in the plasma, as well as to the clinical course, mini-mental state examination (MMSE) score (Folstein et al., 1975), clinical dementia rating (CDR) (Hughes et al., 1982) and apoE phenotype of AD subjects.

were diagnosed with peripheral neuropathy, four with corticobasal degeneration, three with motor neuron disease including amyotrophic lateral sclerosis, two with muscle pain, two with multiple system atrophy and one each with epilepsy, meningitis, myasthenia gravis, multiple sclerosis, cerebral infarction and hepatic encephalopathy.

Materials and methods

ApoE phenotype

AD subjects

ApoE phenotype was determined for all AD subjects after written informed consent was obtained from the patient or a family member. The phenotype was determined from serum samples by the isoelectric focusing and immunoblotting techniques using commercial antibodies (Incstar, Stillwater, MN), as described by Kataoka et al. (1994) previously.

We obtained specimens from 15 Japanese women and 8 Japanese men ranging in age from 60 to 82 with a median age of 69.8 years, who visited our clinics in 2000–2003 without selection if their plasma and CSF were available. These subjects fulfilled the AD criteria of Diagnostic and Statistical Manual-IV and the NINCDS-ADRDA criteria (McKhann et al., 1984). Subjects with a genetic-linkage were excluded. Subjects with mild cognitive impairment (CDR = 0.5) were included when they later fulfilled the AD criteria after progression. The clinical course was defined as the period from the onset to the lumbar puncture. The degree of dementia was expressed by the MMSE score (Folstein et al., 1975) and the severity by CDR (Hughes et al., 1982). Non-AD subjects We obtained specimens from 11 Japanese women and 11 Japanese men ranging in age from 60 to 79 with a median age of 70.2 years, who visited our clinics in 2000–2003 without selection if their CSF and plasma were available. Non-AD subjects refer to neurological non-AD subjects. Five subjects

CTL subjects We obtained specimens from 14 Japanese healthy women and 7 Japanese healthy men ranging in age from 62 to 80 with a median age of 68.5 years, who underwent physical and neuropsychological examinations and replied to the questionnaire for clinical information without selection if their plasma were available. All control subjects fulfilled the “healthy” criteria that were no history of major neuropsychological systemic disorders, normal findings on physical and neurological examinations (MMSE score ≥28), and normal findings on brain MRI. Plasma and CSF from AD, non-AD and CTL subjects This study was approved by the human subjects committee of our university hospital. CSF of AD and non-AD subjects was taken by routine lumbar puncture within 1 month of first consult, after written informed consent was obtained from the patient or a family member. Similarly, plasma of AD, non-AD and CTL subjects was taken. Plasma and CSF specimens were collected and centrifuged at 3000 rpm and 1500 rpm, respectively, for 10 min and aliquoted and stored at −80 °C until analysis.

Enzyme-linked immunosorbent assay of Aβ(1–42) The levels of Aβ(1–42) in plasma and CSF were determined by a sandwich enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody (Mab), 21F12, which was specific for the C-terminus of Aβ(1–42) as the capturing agent, and a biotinylated monoclonal anti-Aβ(1–42) N-terminal antibody, 3D6 for detection (INNOTEST β-amyloid(1–42); Innogenetics, Gent, Belgium) (Andreasen et al., 1999; Vanderstichele et al., 2000). It did not cross-react with Aβ(1–40). The plasma or CSF samples and the standards were assayed in duplicate. Polymerization assay Aβ(1–40) (lot number 530108, Peptide Institute, Inc., Osaka, Japan) and Aβ(1–42) (lot number 521205, Peptide

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Institute) were dissolved by briefly vortexing in a 0.02% ammonia solution at a concentration of 500 μM (2.2 mg/mL) and 250 μM, respectively, in a 4°C room. The supernatant after centrifugation at 4°C for 30 min at 1.6 × 104 × g was stored at −80°C before assaying (fresh Aβ(1–40) and Aβ(1–42) solutions). The polymerization assay was performed as described elsewhere (Naiki et al., 1997). Briefly, the reaction mixture contained 50 μM Aβ(1–40) or 25 μM Aβ(1–42), 50 mM phosphate buffer, pH 7.5, 100 mM NaCl and 0 or 1% (vol/vol) plasma or 0 or 78% (vol/vol) CSF. Thirty-microliter aliquots of the mixture were put into oil-free PCR tubes (size; 0.5 mL, code number; 9046, Takara Shuzo Co. Ltd., Otsu, Japan). The reaction tubes were put into a DNA thermal cycler (PJ480, Perkin Elmer Cetus, Emeryville, California). Starting at 4°C, the plate temperature was elevated at maximal speed to 37°C. Then, they were incubated for 0–9 days as indicated in each figure, and the reaction was stopped by placing the tubes on ice. The reaction tubes were not agitated. From each reaction tube, triplicate 5-μL aliquots were removed then subjected to fluorescence spectroscopy and the mean of each triplicate determined.

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cients to determine the relationship between the final levels of fAβ formation in plasma and the final levels of fAβ formation in CSF, those in plasma, the levels of Aβ(1–42) in CSF. The association between the final levels of fAβ formation in plasma and CDR was analyzed with the Kruskal–Wallis test. A P value less than 0.05 was considered significant. Results Inhibitory effects of plasma obtained from AD, non-AD and CTL subjects on the formation of fAβ from fresh Aβ As shown in Figs. 1A and B, the fluorescence of ThT after incubation of fresh Aβ(1–40) or Aβ(1–42) at 37°C showed a characteristic sigmoidal curve. This curve is consistent with the nucleation-dependent polymerization model (Naiki et al., 1997). fAβ(1–40) and fAβ(1–42) stained with Congo red showed typical orange-green birefringence under polarized light (data not shown). The plasma obtained from all AD, nonAD and CTL subjects decreased the final levels of fAβ(1–40) formation from fresh Aβ(1–40) (Fig. 1A). A similar inhibitory effect of plasma was observed on fAβ(1–42) formation (Fig.

Fluorescence spectroscopy, electron microscopy and polarized light microscopy A fluorescence spectroscopic study was performed on a Hitachi F-2500 fluorescence spectrophotometer as described elsewhere (Naiki and Nakakuki, 1996). Optimum fluorescence of fAβ(1–40) and fAβ(1–42) was measured at the excitation and emission wavelengths of 445 and 490 nm, respectively, with the reaction mixture containing 5 μM thioflavin T (ThT) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 50 mM of glycine–NaOH buffer, pH 8.5. Electron microscopy and polarized light microscopy of the reaction mixtures were performed as described elsewhere (Hasegawa et al., 1999). Other analytical procedures The Alb concentration in plasma was measured by using the color reaction with bromcresol green (Sysmex Co., Kobe, Japan) (Doumas et al., 1971). The IgG level in plasma was quantified by the fixed-time nephelometry on the BN 2 (Dade Behring Marburg, GmbH) (Bossuyt and Blanckaert, 1999). Statistical analysis The final levels of ThT fluorescence in AD, non-AD and CTL subjects in the study population were analyzed by nonparametric analysis based on the Kruskal–Wallis test. Post hoc comparisons were made using the Tukey–Kramer procedure. Multiple comparisons are made between the final levels of fAβ formation in plasma, the final levels of fAβ formation in CSF, the levels of Aβ(1–42) in CSF, the levels of Aβ(1–42), Alb and IgG in plasma, MMSE, clinical course and apoE phenotype by stepwise regression. Correlations were analyzed according to Pearson's correlation test using Pearson's correlation coeffi-

Fig. 1. Effects of the plasma obtained from AD, non-AD and CTL subjects on the kinetics of fAβ(1–40) (A) and fAβ(1–42) formation (B) from fresh Aβ (1–40) and Aβ(1–42), respectively. The reaction mixtures containing 50 μM Aβ(1–40) (A) or 25 μM Aβ(1–42) (B), 50 mM phosphate buffer, pH 7.5, 100 mM NaCl and 0 (●) (n = 8) or 1% (vol/vol) plasma obtained from AD (○) (n = 23), non-AD (n) (n = 22) or CTL subjects (□) (n = 21) were incubated at 37°C for the indicated times. Each symbol represents the mean value. At all points, standard errors are within the diameter of symbols. Each figure is a representative pattern of 3 independent experiments.

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1B). Moreover, the plasma from non-AD (non-AD-plasma) and CTL subjects (CTL-plasma) inhibited the formation of both fAβs more strongly than did the AD-plasma (Figs. 1A and B). We confirmed that the reaction mixtures containing AD-, nonAD- or CTL-plasma with no Aβs exhibited no significant ThT fluorescence (0.02 ± 0.02 vs. 0.03 ± 0.01 vs. 0.04 ± 0.01 for AD-, non-AD- and CTL-plasma, respectively; mean ± SE, n = 3 each, the same arbitrary unit as in Fig. 1). As shown in Fig. 2A, incubation with AD-plasma gave a significantly higher final level of fAβ(1–40) formation than that with non-AD-plasma (1.72 ± 0.28 vs. 0.96 ± 0.11 (mean ± SE), P < 0.05) and CTL-plasma (1.72 ± 0.28 vs. 0.40 ± 0.04 (mean ± SE), P < 0.01). Similarly, incubation with AD-plasma gave a significantly higher final level of fAβ(1–42) formation than that with non-AD-plasma (4.65 ± 0.35 vs. 3.67 ± 0.28, P < 0.05) and CTL-plasma (4.65 ± 0.35 vs. 3.41 ± 0.08 (mean ± SE), P < 0.01) (Fig. 2B). In both cases, incubation with AD-plasma gave significantly lower final levels of fAβ(1–40) and fAβ(1–42) formation than the incubation without plasma (1.72 ± 0.28 vs. 8.94 ± 0.17 in fAβ(1–40) formation, 4.65 ± 0.35 vs. 11.44 ± 0.28 in fAβ(1–42) formation, P < 0.001 in both cases). The comparison of final level between non-AD- and CTL-plasma did not show a significant difference. Incubation of fresh Aβ(1–40) without plasma gave clear fibril formation electron-microscopically (Fig. 3A). The fAβ formed from fresh Aβ(1–40) assumed the nonbranched, helical

filament structure of approximately 7 nm in width and exhibited a helical periodicity of approximately 220 nm, as described previously (Naiki and Nakakuki, 1996). Protofibrils with a diameter of about 4 nm (Harper et al., 1997; Harper et al., 1999) were not observed in the reaction mixture. On the other hand, many shorter, sheared fibrils were observed after incubation with the plasma obtained from an AD subject (Figs. 2 and 3B). Similar morphology was observed after the incubation with the plasma obtained from 2 other AD subjects. Fibrils were rarely found, and small amorphous aggregates were occasionally observed after incubation of fresh Aβ(1–40) with the plasma obtained from a non-AD subject (Figs. 2 and 3C). Similar morphology was observed after the incubation with the plasma obtained from 2 other non-AD and 3 CTL subjects. We also obtained morphological findings after incubation of Aβ(1–42) with AD-, non-AD- and CTL-plasma similar to Aβ(1–40) (data not shown). The control plasma samples without Aβs contained fine round to polygonal particles with a diameter ranging from 20 to 80 nm, as well as occasional large aggregates with a diameter ranging from 100 to 250 nm (Fig. 3D). Similar morphology was observed with AD-, non-AD- and CTL-plasma. Both fAβ(1–40) and fAβ(1–42) formed by incubation with non-AD- and CTLplasma were stained with Congo red to a lesser degree than those formed by incubation with AD-plasma (data not shown). These data indicate that this ThT assay reliably measures the formation of mature amyloid fibrils in our experimental conditions. However, we cannot rule out the possibility that Aβ protofibrils and other forms of Aβ aggregates are formed in the reaction mixture and that the ThT assay also detects them significantly. Multiple comparisons between the final levels of fAβ formation after incubation with plasma, the final levels of fAβ formation after incubation with CSF, the levels of Aβ(1–42) in CSF, the levels of Aβ(1–42), Alb or IgG in plasma, MMSE, clinical course and apoE phenotype of AD subjects In multiple comparisons between the final levels of fAβ(1–40) and fAβ(1–42) formation after incubation with AD-plasma, the final levels of fAβ formation after incubation with CSF, the levels of Aβ(1–42) in CSF, the levels of Aβ(1–42), Alb or IgG in plasma, MMSE, clinical course and the number (i.e., 0, 1 or 2) of E4 isoform of apoE of AD subjects, only the levels of Aβ(1–42) in CSF were associated with the final levels of fAβ(1–42) formation after incubation with AD-plasma significantly (F = 5.427, P < 0.05).

Fig. 2. Scatter plots of the final levels of fAβ(1–40) (A) and fAβ(1–42) formation (B) incubated with AD-, non-AD- or CTL-plasma. ThT fluorescence 9 days (A) or 24 h (B) after the initiation of the reaction was plotted for AD (n = 23), non-AD (n = 22) and CTL (n = 21) subjects. The plasma obtained from 3 AD (○), 3 non-AD (○) and 3 CTL subjects (○) was further analyzed by electron microscopy (see Fig. 3). Mean values ± SE are shown to the right of the individual data for each group. *P < 0.05, **P < 0.01, Tukey–Kramer procedure.

Comparisons between the final levels of fAβ formation after incubation with AD-plasma and the final levels of fAβ formation after incubation with AD-CSF The final levels of fAβ(1–40) and fAβ(1–42) formation in AD-plasma increased with the increase in the final levels of fAβs formation in CSF from AD-subjects (AD-CSF), but not significantly (r = 0.401, P = 0.059 in fAβ(1–40) formation, r = 0.411, p = 0.053 in fAβ(1–42) formation) (Fig. 4A and data not shown).

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Fig. 3. Electron micrographs of fAβ(1–40) formed with or without plasma and the control plasma sample without Aβs. The reaction mixtures containing 50 μM Aβ(1–40), 50 mM phosphate buffer, pH 7.5, 100 mM NaCl and 0 (A) or 1% (vol/vol) plasma obtained from an AD (B) or a non-AD subject (C) were incubated at 37°C for 9 days. Similar morphology was observed with the CSF of 2 other AD and 2 other non-AD and 3 CTL subjects (data not shown). See the legend to Fig. 2. The control mixture containing 50 mM phosphate buffer, pH 7.5, 100 mM NaCl and 1% (vol/vol) plasma obtained from an AD subject (D) was also examined. Similar morphology was observed with the CSF of 2 other AD, 3 other non-AD and 3 other CTL subjects (data not shown). Scale bars indicate a length of 250 nm.

Comparisons between the final levels of fAβ formation after incubation with AD-plasma and the concentrations of Aβ(1–42) in CSF or those in plasma As shown in Fig. 4B, the final levels of fAβ(1–42) formation after incubation with AD-plasma showed a significant negative correlation with the concentration of Aβ(1–42) in CSF (r = 0.453, P < 0.05 in Fig. 4B), although the final levels of fAβ(1–40) formation did not. On the other hand, the final levels of fAβ(1–40) and fAβ(1–42) formation after incubation with AD-plasma showed no significant correlation with the concentration of Aβ(1–42) in plasma. Association between the final levels of fAβ formation after incubation with AD-plasma and CDR of AD subjects The final level of fAβ(1–40) and fAβ(1–42) formation after incubation with AD-plasma was not associated with CDR significantly (data not shown). Discussion Recently, we found that the CSF of both non-AD subjects (non-AD-CSF) and AD subjects inhibited the formation of fAβ(1–40) and fAβ(1–42), but the non-AD-CSF inhibited the formation more strongly (Ono et al., 2005). In AD subjects, the final levels of fAβ(1–40) and fAβ(1–42) formation showed a significant negative correlation with the Aβ(1–42) level in CSF (Ono et al., 2005). In the present study, we demonstrated that human plasma also inhibited the formation of fAβ(1–40) and

fAβ(1–42). Moreover, non-AD-plasma (n = 22) and CTLplasma (n = 21) inhibited the formation of fAβs significantly more strongly than AD-plasma (n = 23). The weaker inhibitory activity in the plasma of AD subjects may be due to the decrease in specific inhibitors of fAβ formation. Previous studies including ours showed that various molecules in plasma and CSF, such as apolipoprotein E (apoE) (Naiki et al., 1997), serum amyloid P component (SAP) (Janciauskiene et al., 1995), α1-antichymotrypsin (ACT) (Eriksson et al., 1995) and α2-macroglobulin (α2M) (Du et al., 1998) inhibited fAβ formation in vitro. Recently, it was reported that naturally occurring human anti-Aβ antibodies block fAβ formation and prevent Aβ-induced neurotoxicity in vitro (Du et al., 2003). Conflicting data have been obtained in previous studies on these concentrations in the blood probably because the source of these factors in blood in AD is unknown. The mean plasma concentrations of α2M did not significantly differ between AD subjects and controls (Scacchi et al., 2002). SAP levels in sera of AD subjects were reported to be significantly lower than that in the control group (Nishiyama et al., 1996). Naturally occurring human anti-Aβ antibodies have been detected in the blood as well as CSF, but there was no difference between the healthy elderly and AD subjects (Hyman et al., 2001). In our study, the final level of fAβs after incubation with AD-plasma was not significantly correlated with the concentrations of Alb and IgG in AD-plasma. However, as apoE phenotyping in AD subjects was limited to a small number in our study, the correlation needs to be

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Fig. 4. Correlation of the final levels of fAβ(1–42) incubated with AD-plasma, with the final levels of fAβ(1–42) incubated with AD-CSF (A) and with the concentration of Aβ(1–42) in CSF (B). ThT fluorescence 24 h after the initiation of the reaction was plotted against the final levels of fAβ(1–42) incubated with AD-CSF (A) or against the concentration of Aβ(1–42) in CSF (B). In A, the final level of fAβ(1–42) incubated with AD-plasma increased with the increase in the final level of fAβ(1–42) incubated with AD-CSF, but not significantly (r = 0.411, P = 0.053 in A; Pearson's correlation test). In B, a significant negative correlation was observed between the final levels and Aβ(1–42) concentration (r = 0.453, P < 0.05 in B; Pearson's correlation test).

studied further with a larger number of samples. Thus, a particular set of the above-described molecules may contribute additively or synergistically to inhibit fAβ formation in vitro and in vivo. Another possibility is that specific factors accelerating fAβ formation such as apoE and ACT that each recognize and bind to Aβ to promote filament formation (Ma et al., 1994) may be increased in the plasma as well as the CSF of AD subjects. The blood and CSF levels of ACT have been reported to be increased (Matsubara et al., 1990) or normal (Pirttila et al., 1994) in AD. Recently, it was reported that ACT levels were elevated in plasma and CSF of AD subjects and the levels increased with progression of AD by measuring ACT concentration in a large number of carefully studied subjects (DeKosky et al., 2003). Moreover, it was reported a significant positive relationship between CSF and plasma levels of ACT (Sun et al., 2003). It was reported a significant increase in the levels of plasma apoE in non-fasted late-onset AD subjects

when compared with the levels of control individuals (Taddei et al., 1997), while some groups reported that the apoE level is not increased in AD (Scacchi et al., 1999). In some clinical situations, apoE and ACT may also act as a promoter of fAβ formation. Although some inhibitors and accelerators in plasma as well as CSF have been studied previously, we provided the first evidence that total balance of these factors in plasma is significantly different between AD and non-AD subjects by examining the influence of plasma on the formation of fAβ(1– 40) and fAβ(1–42) in vitro. Further investigation is necessary including the association between the degree of fAβ formation and concentrations of candidate molecules, which are known or newly identified by a proteome analysis of AD- and nonAD-plasma and CSF. In our study, the final levels of fAβ(1–40) and fAβ(1–42) formation in plasma were increased with the increase in the final level of fAβs formed in CSF, although not significantly (Fig. 4A). Additionally, the final level of fAβ(1–42) formation after incubation with AD-plasma showed a significant negative correlation with the concentration of Aβ(1–42) in CSF (Fig. 4B). We found that fAβ(1–40) and fAβ(1–42) formation after incubation with AD-CSF showed a significant negative correlation with the concentration of Aβ(1–42) in CSF and that the final level of fAβ(1–42) formation after incubation with AD-CSF also showed a significant positive correlation with CDR (Ono et al., 2005). It was reported that the Aβ(1–42) level in AD-CSF declines with the progression of AD (Jensen et al., 1999). Moreover, a recent populationbased autopsy study found a strong association between lower Aβ(1–42) in CSF levels and high numbers of plaques in the neocortex and hippocampus (Strozyk et al., 2003). Overall, our correlation analysis indicates that the final levels of fAβ(1–42) formation after incubation with AD-plasma as well as AD-CSF may increase in parallel with the progression of AD and fAβ formation in plaques, suggesting that the decrease in CSF Aβ(1–42) level in AD may be due to, at least in part, to fAβ formation in plaques. On the other hand, the final levels of fAβ(1–40) and fAβ(1–42) formation after incubation with AD-plasma showed no significant correlation with the concentration of Aβ(1–42) in plasma. The concentrations of Aβ(1–40) and Aβ(1–42) in plasma have little or no association with the progression of AD (Mayeux et al., 2003). Thus, the plasma Aβ(1–40) and Aβ(1–42) levels may not be influenced by the increase of fAβ(1–40) and fAβ(1– 42) formation in AD-plasma. We speculate that, since the Aβ concentration in AD-plasma is significantly lower than that in AD-CSF (Mehta et al., 2001), the favorable environment for fAβ formation in AD-plasma cannot cause Aβ to selfaggregate and form fAβ extracellularly. Our speculation is not always consistent with previous implications that Aβ deposition may occur in multiple sites throughout the body in AD subjects (Heinonen et al., 1995; Joachim et al., 1989; Soininen et al., 1992). If the Aβ concentration reaches a level high enough to self-aggregate and form fAβ in the environment in contact with plasma, fAβ deposition may occur in systemic organs or tissues in some AD subjects.

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