Coordinated increase of γ-secretase reaction products in the plasma of some female Japanese sporadic Alzheimer's disease patients: quantitative analysis of p3-Alcα with a new ELISA system
- Tomoko Konno†1,
- Saori Hata†1,
- Yukiko Hamada2,
- Yuko Horikoshi-Sakuraba2,
- Tadashi Nakaya1,
- Yuhki Saito1,
- Tohru Yamamoto1,
- Takayuki Yamamoto3,
- Masahiro Maeda2,
- Takeshi Ikeuchi4,
- Sam Gandy†5, 6, 7,
- Hiroyasu Akatsu†3,
- Toshiharu Suzuki†1Email author and
- the Japanese Alzheimer's Disease Neuroimaging Initiative
© Konno et al; licensee BioMed Central Ltd. 2011
Received: 12 July 2011
Accepted: 8 November 2011
Published: 8 November 2011
Aggregatable amyloid β-peptide (Aβ) and non-aggregatable p3-Alcα are metabolic products of the γ-secretase cleavage of amyloid β-protein precursor (APP) and Alcadeinα (Alcα), respectively. Familial AD (FAD) -linked mutations in the presenilin 1 or 2 (PS1 or PS2) component of γ-secretase can cause alternative intramembranous processing of APP and Alcα, leading to a coordinated generation of variants of both Aβ and p3-Alcα. Variant Alcα peptides have been observed in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment and sporadic Alzheimer's disease (AD). Since, like APP, Alcα is largely expressed in brain, one might predict that alternative processing of Alcα would be reflected in body fluids of some AD patients. These patients with misprocessing of multiple γ-secretase substrates might define an endophenotype of p3-Alcα, in whom AD is due either to dysfunction of γ-secretase or to a disorder of the clearance of hydrophobic peptides such as those derived from transmembrane domains.
We developed a simple procedure for extraction of p3-Alcα from plasma and for analyzing this extract in a sensitive, p3-Alcα-specific sandwich enzyme-linked immunosorbent assay (ELISA) system. Plasma p3-Alcα levels and Aβ40 levels were examined in sporadic AD subjects from two independent Japanese cohorts. In some of these patients, levels of plasma p3-Alcα were significantly higher, and were accompanied by parallel changes in Aβ40 levels. This AD-related difference was more marked in female subjects, but this phenomenon was not observed in subjects with frontotemporal lobar degeneration (FTLD).
Reagents and procedures have been established that enable extraction of p3-Alcα from plasma and for quantification of plasma p3-Alcα levels by ELISA. Some populations of AD subjects apparently show increased levels of both p3-Alcα and Aβ40. Quantification of p3-Alcα level may be useful as a readily accessible biomarker for a population of sporadic AD patients in which disease pathogenesis is associated with either dysfunction of γ-secretase or with a disorder of the clearance of transmembrane domain-derived peptides.
Alcadein proteins (Alcs) represent a family of neuronal type I transmembrane proteins composed of Alcadeinα (Alcα), Alcadeinβ (Alcβ) and Alcadeinγ (Alcγ). The Alcs are encoded by three independent genes that are highly conserved among mammals, as is the gene encoding the Alzheimer's amyloid β-protein precursor (APP). Alcs and APP are largely co-localized in brain neurons where they are also frequently cross-linked by the X11L adaptor. Both Alc and APP accumulate in the dystrophic neurites surrounding senile plaques of Alzheimer's disease (AD) brain and participate in the molecular pathobiology of the disease [1–5]. Although Alcs undergo alternative processing in sporadic AD , our data indicate that Alcs are not aggregatable (unpublished observations), and Alcs do not appear in amyloid deposits .
Both APP and the Alcs are substrates for primary proteolytic cleavage by the APP α-secretases ADAM10 and ADAM17. These cleavages are directed toward the holoproteins within their respective intralumenal juxtamembrane domains, causing shedding of the ectodomains and retention of membrane-bound carboxyl terminal fragments (CTFs) . Next, the identical γ-secretase can cleave either the APP-CTF or the Alc-CTF within the transmembrane region. This sequential α- and γ-cleavage of APP yields the p3 peptide, and, by analogy, the α- and γ-cleavage of Alc generates the p3-Alc peptide (amino acid sequence of p3-Alcα along with Aβ and p3 are shown in Additional file 1, Figure S1A).
A substantial body of research has established that pathogenic mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes underlie most familial Alzhemer's disease (FAD: ), and that PS1 or PS2 forms the catalytic site of the γ-secretase aspartyl proteinase complex . There are ~200 known pathogenic PS1/PS2 mutations, and all are believed to act by shifting registration of the intramembranous cleavage of APP within the catalytic site of γ-secretase so as to increase the ratio of the level of the minor and more aggregation prone Aβ species (Aβ42) relative to that of the major and less aggregatable species (Aβ40) (also known as the Aβ42/40 ratio; [8, 9]). While there is controversy around whether PS mutations act by elevating Aβ42, by decreasing Aβ40, or both, there is consensus around the concept that increases in the Aβ42/40 ratio are believed to lead to AD by permitting the formation of neurotoxic Aβ oligomer(s) [10, 11].
Recently, low cerebrospinal fluid (CSF) Aβ42 [12, 13] and low plasma Aβ42/40 [14–16] have been identified as possible biomarkers for AD. Aβ42 levels are determined by a composite of Aβ42 generation, aggregation, and deposition. In order to study the role of γ-secretase catalysis apart from the effects of Aβ42 aggregation, we [4, 5] and others  have studied metabolism of non-APP-derived, nonamyloidogenic γ-secretase reaction products. Using a cell culture model, we have demonstrated that Alcα cleavage by γ-secretase is modulated by a panel of FAD-linked PS1 mutations, all of which increased the proportionate representation of minor p3-Alcα species with variant C-termini . Thus, we have proposed that levels and/or ratios of p3-Alcα species in CSF may have clinical utility as surrogate markers of γ-secretase dysfunction . Conceivably, patients with AD due to γ-secretase dysfunction might be most likely to respond to γ-secretase modulator therapies.
Reduction in clearance of CSF Aβ has recently been associated with sporadic AD (SAD; ), a phenomenon that might well be explained by Aβ aggregation or oligomerization, since there is clear evidence that aggregated Aβ is cleared more slowly than non-aggregated forms. A less likely, but not impossible, scenario that could lead to elevated levels of Aβ and p3-Alc species involves a formulation wherein SAD might, in some cases, be attributable to a defect in clearance of transmembrane (TM) domain-derived peptides. This formulation would dovetail well with evidence that apolipoprotein E (APOE) isotype modulates Aβ clearance [19, 20] and raises the possibility that APOE genotype may modulate clearance of many TM domain-derived peptides, including Aβ and p3-Alc. In separate work still in progress, we are studying whether there exists any relationship between APOE genotype in the levels of plasma and CSF p3-Alc. In any event, there is substantial support for the possibility that alterations in Aβ generation and/or clearance are considered to be possible underpinnings for at least some cases of SAD. These alternative hypotheses are not mutually exclusive and could underlie different biochemical endophenotypes that all lead to clinical AD.
Here we report the establishment of the reagents and procedures that will enable the extraction of p3-Alcα from plasma and the quantification of plasma p3-Alcα levels by ELISA. We also investigate the possibility that quantification of plasma levels of both p3-Alcα and Aβ40 may be useful as readily accessible biomarkers for defining endophenotypes of sporadic AD that are associated with either a dysfunction of γ-secretase or with a disorder of the clearance of TM domain-derived peptides.
Extraction and quantification of serum and plasma p3-Alcs
Supplementary Figure S1A displays the amino acid sequences and cleavage sites of human p3-Alcα and Aβ. Non-aggregatable p3-Alc peptides are detectable in CSF , and changes in their quality and quantity have been proposed to represent evidence for γ-secretase dysfunction in SAD (; Hata et alsubmitted for publication). Because circulating biomarkers offer advantages over CSF biomarkers for noninvasive and/or serial sampling, we sought to detect p3-Alcα in serum and plasma.
In a pilot study, efforts focused on detecting endogenous p3-Alcα in serum. Healthy human serum (HS), newborn calf serum (CS), fetal bovine serum (FBS) and bovine serum albumin solution (BSA, 10 mg/mL in PBS), or these same preparations containing synthetic p3-Alcα35 (10 ng) were subjected to immunoprecipitation after samples were depleted of immunoglobulins by pre-clearing with protein-G Sepharose beads (GE Healthcare). The immunoprecipitates were probed by Western blotting. Endogenous p3-Alcα was detected in FBS, while it was not detected in HS, CS, or the control BSA solution. These data indicate that, at least sometimes, endogenous p3-Alcα is detectable in serum (Additional file 1, Figure S1B).
Although endogenous p3-Alcα was detectable in serum, this fluid contained factor(s) that interfered with the immunodetection of p3-Alcα (Additional file 1, Figure S1B). When serum spiked with synthetic p3-Alcα35 peptide (10 ng) was analyzed, a strong signal was detected in the FBS and BSA solutions. However, p3-Alcα35 peptide was not initially detectable in HS or CS even after spiking with synthetic p3-Alcα35 peptide, although overexposure of the film revealed a weak signal in HS (Additional file 1, Figure S1B). Therefore, this pilot study suggested that; (i) endogenous p3-Alc is present in serum, but that (ii) serum contains factor(s) other than IgG that interfere with one or more steps in the immunoprecipitation/immunodetection protocol for detecting either endogenous or exogenous p3-Alcα.
This result confirmed that both adult serum and plasma contained factor(s) that interfered with the determination of p3-Alcα by ELISA even following a four-fold dilution (Figure 2A). This suggested to us that chemical separation of p3-Alc with quantitative recovery would be necessary to remove the interfering factor(s), and therefore to optimize the assay of p3-Alc in plasma or serum. A standard organic extraction protocol (chloroform:methanol 2:1) was used to recover synthetic p3-Alcα35 peptide from neat samples of healthy human serum (Figure 2B, left, squares) and plasma (Figure 2B, right, squares). After using this extraction protocol to pre-process samples, we observed a standard curve whose slope was almost identical to that of the standard curve in which identical amounts of the synthetic p3-Alcα35 peptide were dissolved in buffer A (closed diamonds). This indicated that factor(s) interfering with the immunoreaction were largely excluded by the chloroform:methanol extraction (see Materials and Methods). This procedure resulted in yields (according to ELISA) that were 82% for peptide (625 pg/mL) dissolved in serum (left) and 91% for that dissolved in plasma (right) (Figure 2B), relative to the identical concentration of peptide dissolved in buffer A. Recovery of p3-Alcα35 from plasma via the extraction protocol was high enough (> 90%) to be considered quantitative.
p3-Alcα levels in serum and plasma derived from the same subjects.
Mean ± SD
Mean ± SD
129 ± 3
148 ± 6
103 ± 8
154 ± 15
142 ± 2
165 ± 0
151 ± 24
178 ± 13
182 ± 12
245 ± 10
Plasma p3-Alcαlevels in AD and FTLD subjects (Japanese cohort 1)
Summary of subject information (Japanese cohort 1)
75 ± 7
197 ± 50
335 ± 82
26 ± 7
76 ± 6
193 ± 57
342 ± 77
28 ± 8
75 ± 7
200 ± 47
331 ± 86
25 ± 7
64 ± 12
190 ± 31
366 ± 51
30 ± 6
65 ± 14
197 ± 20
371 ± 57
29 ± 7
61 ± 9
183 ± 41
361 ± 47
31 ± 7
Despite the fact that FTLD serves as a useful "other neurological disease" (OND) control, Japanese cohort 1 does not include non-demented subjects, and FTLD subjects do not match AD subjects with regard to average age (Table 2), therefore, we next examined plasma p3-Alcα levels in Japanese cohort 2 which included age-matched nondemented controls.
Plasma p3-Alcαlevels in AD and non-AD subjects (Japanese cohort 2)
Summary of subject information (Japanese cohort 2)
76 ± 7
232 ± 59
378 ± 113
74 ± 7
212 ± 54
366 ± 143
78 ± 7
245 ± 58
386 ± 92
73 ± 6
163 ± 70
254 ± 63
71 ± 7
205 ± 19
267 ± 49
75 ± 6
142 ± 77
247 ± 69
Unextracted plasma samples from this cohort were also studied. Aβ40 levels in AD subjects distributed within a higher range than that observed for age-matched normal controls (Figure 4B, upper). The higher plasma Aβ40 levels in female, but not male, AD subjects were readily obvious to visual inspection (Figure 4B, middle, lower). Levels of p3-Alcα and Aβ40 were increased in parallel in AD subjects and were higher than the levels observed in elderly normal controls.
Correlation between levels of p3-Alcαand Aβ40
Analysis of levels of p3-Alcα and Aβ in samples from normal subjects revealed that there was also a strong correlation in normal subjects (n = 21) between levels of these two peptides (Figure 6B, upper; r = 0.6275, P = 0.0023). The correlation between levels of p3-Alcα and Aβ in samples from both normal females (n = 14) and those from normal males (n = 7) was statistically significant (for female subjects, Figure 6B, middle; r = 0.6117, P = 0.0201: for male subjects, Figure 6B, lower; r = 0.7857, P = 0.0480). Overall, these analyses indicated that there was a significant correlation between levels of p3-Alcα and those of Aβ40 in the plasma of normal subjects, and that this correlation was more robust in the plasma of female AD subjects.
The pathogenesis of sporadic AD is thought to be genetically heterogeneous. The precise identities and relative importance of the many contributory genes remain unclear, and additional loci have recently been identified . This is in contrast to the situation for FAD in which pathogenic mutations of APP and PS genes cause alterations in Aβ speciation [6, 7].
From the analyses of p3-Alcα levels in CSF, we previously proposed that alternative processing of multiple γ-secretase substrates may occurs in some sporadic AD populations. This earlier study was performed using a combination of immunoprecipitation and MALDI-TOF/MS to isolate and quantify p3-Alc in 300 μL of CSF [4, 5]. Although the procedure showed semi-quantitative accuracy and was effective for testing changes in the abundance of p3-Alc species in the brain, CSF sampling is relatively invasive (when compared with blood sampling) and presents a challenge for large and/or longitudinal studies that involve repeated sampling over time. Therefore, we developed a system by which p3-Alc could be quantified in relatively small plasma samples. The combination of simple p3-Alc extraction and highly selective ELISA enabled the quantification of p3-Alcα in 100 μL of plasma (200 μL in duplicate assay). Successful extraction of p3-Alcα from plasma with chloroform/methanol solvent suggests that the majority of p3-Alcα in plasma may be bound to lipid or lipoproteins. While we have not performed further characterization of p3-Alcα in plasma, apolipoprotein E would be a potential suspect and association could vary by APOE isotype. A study of the effect of this variable in both CSF and plasma samples is underway.
We have not yet developed an ELISA system that can perfectly discriminate levels of each individual p3-Alcα species (e.g., p3-Alcα35, p3-Alcα38). Such an assay requires antibodies against the neoepitopes created when the specific C-termini are generated by γ-cleavage. Since the polyclonal 839 antibody was prepared using the C-terminal amino acid sequence of p3-Alcα35 as antigen, the ELISA used in this study displayed a relative specificity for detecting synthetic p3-Alcα35, but there was also crossreactive detection of p3-Alcα39 synthetic peptide at about 50-60% of the sensitivity that was observed for detection of p3-Alcα35 (Figure 1B).
p3-Alcα35 with a C-terminal Thr851 is the major (~90%) peptide among the p3-Alcα species with various C-termini [4, 5]. Thus, we conclude that the increased level of p3-Alcα in AD subjects is probably largely dependent on an increase of p3-Alcα35, although we cannot yet exclude the possibility that another species such as p3-Alcα38 may be selectively increased in AD subjects . Therefore, in the present study, we could not examine qualitative changes of p3-Alcα species in plasma. However, we have successfully developed a potentially useful ELISA for p3-Alc peptides and demonstrated its utility in the detection and quantification of plasma p3-Alcα peptide. Despite the fact that plasma levels of p3-Alcα are about ten-fold less than the levels in CSF, p3-Alcα levels are readily quantifiable in both body fluids using this novel ELISA (Hata et al., in preparation).
Using the novel ELISA specific for p3-Alcα, we found that plasma p3-Alcα levels were significantly higher in some AD subjects who also showed higher Aβ40 levels. In female subjects, an obvious and statistically significant difference in plasma p3-Alcα levels was evident when AD subjects were compared against age-matched elderly nondemented controls. The explanation for why p3-Alcα levels and Aβ40 levels are higher in some female AD subjects remains unclear. It is worth noting that gonadal hormones are known to regulate generation of Aβ [22–24] as well as transcription of the APOE gene . Similar gender dimorphic results were recently obtained in plasma samples of another independent cohort when the same ELISA system was employed, suggesting that selection bias is probably not the explanation for the sex-related differences (Kamogawa et al., in preparation).
Increased levels of plasma Aβ and p3-Alcα in some AD subjects may suggest a γ-secretase enzymopathy, as proposed in analyses of CSF p3-Alc  and CSF APLP1-28 . However, we cannot exclude the possibility that p3-Alcα clearance may be decreased in AD, especially since evidence for defective Aβ clearance has been presented by other investigators [18, 26]. Analyses of the possible roles for APOE genotype, CSF and plasma apoE levels, and CSF and plasma γ-secretase and p3-Alcα-degrading enzyme activities are all underway. It is worth noting that polymorphisms in APOE  and neprilysin  have been linked to increased risk of chronic traumatic encephalopathy and/or AD following traumatic brain injury , and these identical factors could play similar roles here.
In Japanese cohort 2, we observed higher levels of plasma Aβ40 and p3-Alcα. Since p3-Alcα peptides are not deposited in brain or cerebral vessels, the molecular pathogenesis underlying these changes in CSF and plasma p3-Alc levels remains unknown. Serial studies of both plasma and CSF from the same individuals over time are required in order to elucidate the underpinnings of these changes, including the sexual dimorphism. Serial studies of CSF and plasma p3-Alcα from transgenic animals and from subjects in the National Institute on Aging Alzheimer's Disease Neuroimaging Initiative should help clarify these results, and those studies are also underway.
The p3-Alcα in blood is likely to be derived from largely brain because Alcα expresses predominantly in brain neurons  and transgenic mice expressing human Alcα-CTF driven by a neuron-specific PDGF promoter increased p3-Alcα levels in plasma (Hata and Takei, unpublished observation). Alcadein is almost identical to APP with respect to neural expression, cellular distribution, metabolism, and function [1–4]. Furthermore, we would re-emphasize that p3-Alc does not aggregate and is stable in plasma, both of which are desirable qualities in clinical sample preparation and handling and both of which have complicated analysis of Aβ in bodily fluids. These properties are useful for the development of sensitive procedures for the detection of γ-secretase dysfunction, and p3-Alcα plasma levels may be useful in identifying AD subjects whose clinical phenotype is caused by a functional alteration of γ-secretases and/or by defective clearance of transmembrane domain-derived peptides. This endophenotyping may be important for selecting subjects for trials of γ-secretase modulators or plasminogen activator inhibitor-1 inhibitors [30, 31], respectively.
Materials and methods
Blood collection and processing, and quantification of plasma p3-Alcα
Informed consent for the use of all human plasma and serum in this study was obtained from the patients and/or their families and approved by the appropriate ethical boards at each institution. Alzheimer's disease was clinically diagnosed based on two major criteria; Diagnostic and Statistical Manual of Mental Disorders: 4th Edition (DSM-IV) and National Institute of Neurological and Communicational Disorders and Stroke - Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA). In Japanese cohort 1, the clinical diagnoses of patients with FTLD were made on the basis of established clinical criteria . A compilation of clinical characteristics and data is shown in Table 2. Detailed descriptions of all subjects are shown in Additional files 2, Tables S1 and S2.
In Japanese cohort 2, all subjects with AD are inpatients and showed MMSE scores consistent with a diagnosis of either moderate or severe dementia (CDR 2 or CDR 3). Clinical characteristics and data are shown in Table 3. Detailed descriptions of all subjects including the raw values of p3-Alcα are shown in Additional files 2, Tables S3 and S4.
For plasma samples, blood was collected into tubes containing EDTA and centrifuged. For serum samples, blood was collected into tubes with no EDTA and allowed to coagulate. To extract p3-Alcα peptides, a four-fold volume of organic reagent (800 μL of chloroform: methanol [2:1] mixture) was added to plasma or serum (usually 200 μL for duplicate assay) in a conical tube (1.5 mL), agitated well by vortexing or sonication for 10 s, and then left to stand for 1 h at room temperature (18-23°C). Next, a 160 μL aliquot of distilled water was added, and the samples were mixed by vortexing. The samples were centrifuged at 15,000 rpm for 15 min, and the aqueous phase was recovered and dried using a SpeedVac system. The dried sample was dissolved in 250 μL of PBS containing 1% (w/v) BSA and 0.05% (v/v) Tween-20 (buffer A). The samples (×1) or samples further diluted with buffer A (2- and 4-fold) were used for ELISA. Aliquots of 100 μL were studied in duplicate. Generally the undiluted sample (×1) was examined for quantification.
Antibodies and ELISA system
The major p3-Alcα species, p3-Alcα35, is a peptide that includes the sequence from Ala817 to Thr852 of Alcα1 (for the amino acid sequence of p3-Alcα, see Additional file 1, Figure S1A). Polyclonal rabbit antibodies were raised against a peptide containing the sequence between position 817 and 822 (#817 antibody) and a peptide containing the sequence between position 839 and 852 (#839 antibody), and affinity purified with the each respective peptide antigen resin. The antibody 839 was used to capture p3-Alcα, and horseradish peroxidase-conjugated pan p3-Alcα antibody 817 and tetramethyl benzidine were used to detect the captured p3-Alcα colormetrically at OD450. Synthetic p3-Alcα35 peptide was used as a standard assay protein. The specificity for p3-Alcα in the ELISA was confirmed using synthetic p3-Alcα35 (major species of CSF p3-Alcα), p3-Alcα39 and p3-Alcβ37 peptides [4, 5]. Aβ in neat samples without extraction was quantified with sELISA (Wako Pure Chemical Industries Co Ltd for cohort 1 and IBL Co Ltd for cohort 2).
amyloid β-protein precursor
small peptide generated by serial primary and secondary cleavages of Alc
cerebrospinal fluid: FAD: familial Alzheimer's disease
sporadic Alzheimer's disease
sandwich enzyme-linked immunosorbent assay.
This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), a grant from the Ministry of Health, Labor and Welfare (MHLW), and a grant from the New Energy and Industrial Technology Development Organization (NEDO) in Japan. S.G. was supported by the NIA Alzheimer's Disease Research Center grant P50 05138 to Mary Sano.
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