Journal of Neurochemistry, 2007
doi:10.1111/j.1471-4159.2006.04404.x
Amyloid b peptide ratio 42/40 but not Ab42 correlates with
phospho-Tau in patients with low- and high-CSF Ab40 load
Jens Wiltfang,* Hermann Esselmann*, Mirko Bibl, Michael Hüll,à Harald Hampel,§
Holger Kessler,¶ Lutz Frölich,** Johannes Schröder, Oliver Peters,àà Frank Jessen,§§
Christian Luckhaus,¶¶ Robert Perneczky,*** Holger Jahn, Magdalena Fiszer,*
Juan Manuel Maler,* Rüdiger Zimmermann,* Ralf Bruckmoser,* Johannes Kornhuber* and
Piotr Lewczuk*
*Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Erlangen, Germany
Department of Psychiatry and Psychotherapy, University of Göttingen, Göttingen, Germany
àDepartment of Psychiatry and Psychotherapy, University of Freiburg, Freiburg, Germany
§Department of Psychiatry, Ludwig-Maximilian University, Munich, Germany
¶Department of Psychiatry, Saarland University, Homburg, Germany
**Central Institute of Mental Health, Department of Psychiatry, University of Heidelberg, Mannheim, Germany
Section Geriatric Psychiatry, Heidelberg University, Heidelberg, Germany
ààDepartment of Psychiatry, Charité – University Medicine, Berlin, Germany
§§Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany
¶¶Department of Psychiatry and Psychotherapy, Heinrich Heine – University, Düsseldorf, Düsseldorf, Germany
***Department of Psychiatry and Psychotherapy, Technical University of Munich, Munich, Germany
àààDepartment of Psychiatry and Psychotherapy, University Hospital Hamburg-Eppendorf, Hamburg, Germany
Abstract
Neurochemical dementia diagnostics (NDD) can significantly
improve the clinically based categorization of patients with
early dementia disorders, and the cerebrospinal fluid (CSF)
concentrations of amyloid b peptides ending at the amino acid
position of 42 (Abx-42 and Ab1-42) are widely accepted biomarkers of Alzheimer’s disease (AD). However, in subjects
with constitutively high- or low-CSF concentrations of total Ab
peptides (tAb), the NDD interpretation might lead to erroneous
conclusions as these biomarkers seem to correlate better with
the total Ab load than with the pathological status of a given
patient in such cases. In this multicenter study, we found
significantly increased CSF concentrations of phosphorylated
Tau (pTau181) and total Tau in the group of subjects with high
CSF Abx-40 concentrations and decreased Abx-42/x-40
concentration ratio compared with the group of subjects with
low CSF Abx-40 and normal Ab ratio (p < 0.001 in both
cases). Furthermore, we observed significantly decreased Ab
ratio (p < 0.01) in the group of subjects with APOE e4 allele
compared with the group of subjects without this allele. Surprisingly, patients with low-Abx-40 and the decreased Ab ratio
characterized with decreased pTau181 (p < 0.05), and unaltered total Tau compared with the subjects with high Abx-40
and the Ab ratio in the normal range. We conclude that the
amyloid b concentration ratio should replace the ‘raw’ concentrations of corresponding Ab peptides to improve reliability
of the neurochemical dementia diagnosis.
Keywords: amyloid b, biomarkers, cerebrospinal fluid,
dementias.
J. Neurochem. (2007) 10.1111/j.1471-4159.2006.04404.x
Received June 30, 2006; revised manuscript received November 6, 2006;
accepted November 24, 2006.
Address correspondence and reprint requests to Dr Jens Wiltfang,
Molecular Neurobiology Laboratory, Department of Psychiatry and
Psychotherapy, University of Erlangen-Nuremberg, Schwabachanlage 6,
91054 Erlangen, Germany.
E-mail: [email protected]
Abbreviations used: AD, Alzheimer’s disease; APP, b-amyloid precursor protein; CDR, Clinical Dementia Rating; CSF, cerebrospinal
fluid; MCI, mild cognitive impairment; NDD, Neurochemical dementia
diagnostics.
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2006.04404.x
1
2 J. Wiltfeng et al.
With increasing life expectancy of the population, Alzheimer’s disease (AD) becomes one of the most severe
problems in the industrialized countries. Clinically based
diagnosis of AD carries a severe uncertainty: although its
sensitivity is relatively high (93%), specificity may be
lower, e.g. reported as much as 55% in a multicenter
clinical-autopsy controlled study (Mayeux 1998). In expert
hands, the clinical diagnosis of moderate to severe AD
is predictive of AD pathology in 80–90% of cases,
nevertheless, very early diagnosis of AD, and differential
diagnosis of unusual presentations of patients with dementia remains difficult on clinical grounds. Although it is still
considered incurable, the advent of new promising therapeutic avenues like amyloid b immunization (Hock et al.
2003) calls urgently for an improved early and differential
diagnosis of AD.
Since cerebrospinal fluid (CSF) is in direct contact with the
central nervous system, several potentially promising CSF
biomarkers have been tested alone or in combinations
(Vanmechelen et al. 2001a; Blennow and Hampel 2003;
Hampel et al. 2004a; Wiltfang et al. 2005). Among them,
amyloid b peptides (Ab peptides) and Tau protein(s) fulfill the
criteria for good AD diagnostic tests, as summarized by an
expert review (The Working Group on: ‘Molecular and
Biochemical Markers of Alzheimer’s Disease’’s 1998), which
is not surprising since these factors are directly involved in
pathologic events of the disease, namely deposition of senile
plaques and formation of neurofibrillary tangles.
Amyloid b peptides result from the enzymatic cut of
b-amyloid precursor protein (APP) by two enzymes
known as b- and c-secretases, whereas enzymatic
processing of APP by a-secretase leads to non-amyloidogenic pathway. The CSF concentration of one of the peptides
resulting from this process, namely Ab1-42 is regularly
found decreased to about 50% of the normal value in patients
with AD (Wiltfang et al. 2001, 2002; Lewczuk et al. 2004c).
Sensitivity and specificity of Ab1-42 alone to distinguish AD
from elderly controls were 78% and 81%, respectively, in the
study of Hulstaert et al. (1999), and Galasko et al. (1998)
reported similar figures of 78% and 83% for sensitivity and
specificity, respectively.
The disease-related alterations of either CSF total Ab
peptides (tAb) content or the most abundant species, Ab40
have not been reported (van Gool et al. 1995; Ida et al.
1996; Wiltfang et al. 2001, 2002; Lewczuk et al. 2003,
2004b). However, our preliminary observations suggested
that alterations of Ab1-42 and/or Abx-42 concentrations
might not only result from Alzheimer’s disease pathology,
but may also be related to total Ab peptides concentrations.
In such case, healthy individuals with relatively low tAb
might be misdiagnosed as having ‘pathologically low’ Ab142 and Abx-42 concentrations, and vice versa, AD subjects
with high tAb might be misinterpreted as normal Ab1-42
and/or Abx-42-carriers. To test our working hypothesis that
the Ab ratio compensates for constitutively low or high
CSF amyloid b load, we have measured corresponding
biomarkers in carefully selected patients characterizing with
high- or low-CSF tAb, and correlated them to phosphorylated Tau protein as an AD biomarker independent from
Ab pathology.
Materials and methods
Patients and lumbar punctures
The study was approved by the ethics committees of all the
participating universities. All the patients and/or their relatives have
given informed consent. Twelve German gerontopsychiatric university departments have participated in this study recruiting 312
patients with early dementias and mild cognitive impairment (MCI).
Patients with early Alzheimer’s disease (D-AD) were diagnosed
according to the criteria of ICD-10, and the National Institute of
Neurological and Communicative Disorders and the Stroke-Alzheimer’s Disease and Related Disorders Association (NINCDSADRDA) (McKhann et al. 1984). Patients with other types of
dementias (D-O) fulfilled the criteria of corresponding dementias.
Cognitive dysfunction was assessed with CERAD neuropsychological test battery (Thalmann et al. 1998), WMS-R Logical Memory
(Härting et al. 2000), Trail Making Test (Reitan 1979), and Clock
Drawing Test (Shulman et al. 1986). Functional decline was
assessed using informant questionnaires B-ADL (Hindmarch et al.
1998), and IQCODE (Jorm 1994). Distinction of MCI and dementia
was based on Clinical Dementia Rating (CDR) (Morris 1993). A
diagnosis of MCI corresponded to a CDR score of 0.5, a diagnosis
of mild dementia corresponded to a CDR score of 1.0. Severity of
dementia was graded according to the Mini Mental State Examination (Folstein et al. 1975). Patients with MCI were categorized into
MCI of Alzheimer’s disease (MCI-AD), and MCI of other dementia
types (MCI-O) according to clinical judgement. The clinical data are
presented in the Table 1.
Lumbar punctures have been performed in the sitting position
according to the standardized procedure. After collection of the CSF
for routine diagnosis, additional 4.5 mL of the material have been
sampled into a polypropylene test tube for this study. The CSF has
been centrifuged immediately after the tap (1600 g, 20°C, 15 min),
aliquoted into 16 polypropylene test tubes, and frozen within 30–
40 min after the puncture. The material has never been thawed/
refrozen.
Determination of biomarkers with ELISAs
Amyloid b peptides have been determined with commercially
available ELISAs. Briefly, to measure the CSF concentration of
Table 1.
Clinical characteristic of the patients
Groups
n
Age (average, SD)
Sex (m/w)
D-AD
D-O
MCI-AD
MCI-O
74
28
141
69
71.8
65.2
67.1
60.4
33/41
13/15
84/57
46/23
(8.0)
(12.1)
(7.9)
(8.4)
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2006.04404.x
Ab peptide ratio 42/40 correlates with phospho-Tau 3
amyloid b peptides family ending at the amino acid position of 42
(Abx-42), 50 lL of undiluted CSF from patients or standards
included in the kit (The Genetics Co., Zürich, Switzerland) have
been applied to microtiter plates pre-coated with an antibody
recognizing the N-terminal end of the peptide (WO-2). Following an
overnight incubation at + 4°C and washing steps, detection antibody
G2-13 (isotype, IgG1, j) indirectly linked to an enzyme has been
applied. After incubation and washing, chromogene has been added
to incubate for 15–20 min, and the reaction has been stopped with
sulfuric acid. To measure the concentration of Ab peptides family
ending at the amino acid position of 40 (Abx-40) (assay from The
Genetics Co), a similar procedure has been applied, except that
before application to a microtiter plate, the samples were pre-diluted
1 : 5 with diluting buffer. This assay uses the same capturing
antibody (WO-2), and the detection antibody (G2-10, isotype
IgG2b, j) recognizes specifically the amino acid position of 40.
As both assays are unspecific regarding the N-terminus of the Ab
peptides, we decided to use ‘x’ (Lewczuk et al. 2003). Hence, as a
matter of fact, ‘Abx-42’ consists of a family of peptides beginning at
positions of 1, 2, 3, etc, and ending at the position of 42, and
correspondingly ‘Abx-40’ is a family of peptides beginning at
positions of 1, 2, 3, etc, and ending at the position of 40.
Following the measurements of the Ab peptides concentrations,
the amyloid b peptides concentration ratio has been calculated
(Abx-42/Abx-40).
The concentration of pTau181 has been measured with an ELISA
(Innogenetics, Ghent, Belgium) based on the monoclonal antibody
specifically directed against the epitope in question. The phosphorylated molecules of Tau protein have been captured by the first
antibody, HT7. After an overnight incubation at +4°C with a
biotinylated antibody (AT270) followed by washing steps, the
antigen–antibody complex has been detected by a peroxidase-linked
streptavidin. The color reaction has been stopped with sulfuric acid
after the incubation with the substrate solution. The optical density
has been read at 450 nm without correction.
To measure tTau in CSF, undiluted CSF or standards were applied
to each well of microtiter plate (Innogenetics) simultaneously with
conjugate. After an overnight incubation at 20°C followed by
washing steps, second conjugate was applied to incubate for 30 min.
The wells were then washed and substrate solution was applied. The
reaction was stopped with sulfuric acid, and the color reaction was
read at 450 nm with a correction wavelength set at 620 nm.
Ab-SDS-PAGE/immunoblot
To separate Ab peptides in the CSF, and to measure their total
concentration (tAb), the urea version of the N,N’-bis-(2-hydroxylethyl)-glycine/bis-Tris/Tris/sulfate SDS-PAGE has been used as
described by Wiltfang et al. (1991). Briefly, native CSF (10 lL)
in a sample buffer has been applied to a gel slot. Gels were run
at 20°C for 1 h at a constant current of 24 mA/gel using the
MiniProtean II electrophoresis unit (Bio-Rad, Munich, Germany).
Next, semi-dry Western blotting has been performed according to
the protocol of Wiltfang et al. (1997) using PVDF membranes.
Immunostaining has been performed with monoclonal aminoterminal-selective antibody, 1E8. After the washing step, the
membranes have been incubated with an anti-mouse biotinylated
antibody, washed and horseradish peroxidase-coupled streptavidin
has been added for 1 h. After the final wash, the chemilumines-
cence has been visualized with ECLPlus solution (Amersham
Pharmacia, Uppsala, Sweden) according to the protocol of the
manufacturer, using the CCD camera system (FluorSMax MultiImager; Bio-Rad).
To separate and quantify amyloid b peptides in plasma (n = 28),
a similar protocol was applied with the exception that immunoprecipitation step was performed before applying the material onto the
gels (Lewczuk et al. 2004d).
Statistical analysis
Normality of the distribution of Abx-40 CSF concentrations has
been confirmed by Kolmogorov–Smirnov test. Correlations between
methods have been assessed with Pearson’s correlation rank. A
p < 0.05 has been considered significant. Differences between the
groups have been statistically analyzed with Kruskal-Wallis test.
Results
Correlation between Abx-40 and the total Ab peptides
concentration
Figure 1a presents the correlation between CSF concentrations of Abx-40 as measured with ELISA and tAb as
measured with Ab-SDS-PAGE/immunoblot (n = 20,
R = 0.8, p < 0.001).
Distribution of Abx-40 CSF concentration; high- and
low-Ab carriers
Figure 1b shows the normal distribution of Abx-40 in the
population of all the patients included into this study
(average ± SD, 9426 ± 2713 pg/mL, n = 312). Correspondingly, high Abx-40 CSF load was defined as Abx-40
concentration higher than average + 1 SD (12 139 pg/mL,
n = 50), and low Ab CSF load has been defined as Abx-40
lower than average – 1 SD (6713 pg/mL, n = 50).
Comparison of pTau181 concentrations between groups
of patients with high- and low-Abx-40
For further characterization of the groups, we divided
patients with high- and low-Abx-40 load into these with
Abx-42/x-40 ratio characteristic for AD, and these with the
ratio non-characteristic for AD, using the cutoff from our
previously published study optimized for separation of AD
and non-AD dementia patients (0.11) (Lewczuk et al.
2004c). Comparison of pTau181 and total Tau concentrations
between patients with low Abx-40 and Abx-42/x-40 concentration ratio higher than the cutoff, i.e. untypical for the
diagnosis of AD (n = 31), and the patients with high Abx-40
and Abx-42/x-40 concentration ratio lower than the cutoff,
i.e. typical for AD (n = 28) are presented in the Fig. 2. This
shows that patients with Ab ratio characteristic for AD have
significantly higher pTau181 and total Tau (in both cases,
p < 0.001) concentration compared with these with Ab ratio
untypical for AD.
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2006.04404.x
4 J. Wiltfeng et al.
Fig. 1 The correlation of Abx-40 concentrations (ELISA) with total
Ab-peptides concentration (Ab-SDS-PAGE/immunoblot) (1a), and
normal distribution of Abx-40 in the patients studied (1b).
Interestingly, patients with low-Abx-40 and the decreased
Ab ratio (n = 19) characterized with decreased pTau181
(p < 0.05), and unaltered total Tau compared with the
subjects with high Abx-40 and the Ab ratio in the normal
range (n = 22) (Fig. 3).
Only weak (R < 0.3) correlation was observed between
age of the patients and the biomarkers considered in this
study. There were no statistically significant differences
between biomarkers in the CSF of women and men (data not
shown).
Correlation of the biomarkers with the APOE genotype
of the patients
The correlation between the biomarkers studied and the
APOE genotype is presented in the Fig. 4. We observed
significant decrease of Ab ratio (p < 0.01) in patients with e4
allele of the APOE gene compared with these without e4
allele.
Correlation between Ab peptides concentrations
in the CSF and plasma
In 28 patients, we did not observe statistically significant
correlation between CSF and plasma concentrations of any
Ab peptides of the ‘peptide quintet’ studied here (Ab1-37/38/
Fig. 2 Comparison of pTau181 (2a) and total Tau (2b) concentrations in the group of patients with low Abx-40 and Abx-42/Abx-40
concentration ratio >0.11 (left bars) and patients with high Abx-40 and
Abx-42/Abx-40 concentration ratio <0.11 (right bars). Presented are
medians, interquartile ranges (boxes), and 10–90 percentages (whiskers). Cut off values for pTau181 (60 pg/mL), and total Tau (300 pg/
mL) are presented as dotted lines.
39/40/42) (data not shown); however, weak correlation has
been observed in the case of Ab1-42/1-40 concentration ratio
(R = 0.56, p < 0.01).
Discussion
In this paper, we present the limitations of neurochemical
dementia diagnostics in patients with either high- or lowconcentration of amyloid bx-40 peptide in the CSF based on
a multicenter study.
Although there have been several reports showing that the
total concentration of Ab-peptides in human CSF is not
altered in pathological conditions [(Wiltfang et al. 2001,
2002; Lewczuk et al. 2004c), and reviewed in (Ida et al.
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2006.04404.x
Ab peptide ratio 42/40 correlates with phospho-Tau 5
Fig. 4 Abx-42/Abx-40 concentration ratio in subjects with- (left bar),
and without APOE e4 allele. Presented are medians, interquartile
ranges (boxes), and 10–90 percentages (whiskers).
Fig. 3 Comparison of pTau181 (3a) and total Tau (3b) concentrations in the group of patients with high-Abx-40 and normal Abx-42/
Abx-40 concentration ratio (left bars) and patients with low-Abx-40 and
decreased Abx-42/Abx-40 concentration ratio (i.e. <0.11, right bars).
Presented are medians, interquartile ranges (boxes), and 10–90 percentages (whiskers). Cut off values for pTau181 (60 pg/mL), and total
Tau (300 pg/mL) are presented as dotted lines.
1996)], the issue how the total CSF Ab load influence the
reliability of the neurochemical dementia diagnosis has not
been tested so far. Therefore, we addressed this question by
measuring corresponding Ab biomarkers, and relating them
to the concentration of Abx-40, i.e. the most abundant
peptide in human CSF, hypothesized to represent the total
CSF Ab peptides load. Currently, to our best knowledge, no
high throughput method is available to measure the total Ab
CSF load, and since we observed a good correlation between
Abx-40 (the most predominant Ab peptide species in human
CSF), and the total Ab load as measured with a low
throughput Ab-SDS-PAGE/immunoblot, we hypothesize that
Abx-40 concentration describes well the total Ab CSF
concentration.
As independent biomarker describing the other pathological pathway of AD (i.e. hyperphosphorylation of Tau and
formation of neurofibrillary tangles), we have used the CSF
concentration of Tau phosphorylated at threonine 181 as this
biomarker has been widely accepted as a AD-specific one
(Vanmechelen et al. 2000, 2001b; Itoh et al. 2001; Schonknecht et al. 2003; Hampel et al. 2004b; Lewczuk et al.
2004a). We found significant increase of pTau181 and tTau
in patients with high Abx-40 and pathologic [<0.11,
(Lewczuk et al. 2004c)] Ab ratio as compared to the group
of patients with low Abx-40 and normal Ab ratio. This points
to the fact, that in these groups the Ab ratio corresponds to
the expected alterations of AD biomarkers of the ‘Tau
family’, i.e. total, and hyperphosphorylted Tau.
This in turn could mean that the concentrations of Abx-42
are not only influenced by the presence or absence of AD
pathology but are also determined by the rate of APP
metabolism of yet unknown background. Currently pathomechanisms of such differences in APP processing rate can
only be speculated, but there are evidences that a genetic
background could be responsible for it (Lammich et al.
2004). The observed significant decrease of Abx-42 concentration, and Ab ratio in the group of subjects with APOE e4
allele, as well as significant increase of total Tau concentration in this group is in line with the observation of higher
percentage of e4-positive patients among AD patients
compared with other dementias (Holmes 2002).
Interestingly, in a recent study a cluster of patients was
identified that do not show increased CSF tau levels, while
another clusters had most markedly decreased Ab42 (Iqbal
et al. 2005). Moreover, in vitro studies currently discussed
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2006.04404.x
6 J. Wiltfeng et al.
on the AlzForum (http://www.alzforum.org; Gain or Loss of
Function) confirm that the Ab peptides concentration ratio is
a better biomarker of AD pathology than either of these
peptides alone. Our finding are in line with these studies, and
we believe that these finding might potentially have a strong
impact on the interpretation of NDD outcome, since without
knowing the concentration of total Ab peptides (or Abx-40),
the ‘normal’ and ‘decreased’ Ab peptides ending at the
position of 42 may be easily misinterpreted.
Unexpectedly, we found increased pTau181 and unaltered
total Tau concentration in patients with high-Abx-40
concentration and normal Ab ratio compared with these
with low Abx-40 concentration and normal ratio; however, it
must be stressed that in majority of cases of both groups
pTau181 was in the normal range or only slightly increased
(Lewczuk et al. 2004a). This finding, which certainly
requires further studies, might be explained to some degree
by either extremely high- or extremely low-Abx-42 concentrations in both groups, respectively (considering the mathematical definition of the Abx-42/x-40 concentration ratio).
To our best knowledge, this report presents the distribution
of Abx-40 CSF concentration based on the biggest number
of cases but not healthy controls. Explicit data of the
distribution of Abx-40 in healthy individuals are, to our
knowledge, not available in the literature, however, from the
inspection of the Fig. 4 in the report by Ida et al. (1996), it
can be expected that this distribution is normal, too.
A satisfying explanation of decreased Ab42 species in
CSF of AD is still lacking. The suggested mechanism of
decreased Ab42 concentration in CSF due to a simple
accumulation in plaques is not sufficient, since we found
decreased Ab42 concentrations in CSF of patients with
Creutzfeldt–Jakob disease with no apparent plaques at all
(Wiltfang et al. 2003). This points to other mechanisms, such
as epitope masking, and chaperones dysfunction, and
certainly requires further studies. Similarly, it is still not
clear if increased concentration of the phospho-Tau is due to
increased activity of kinase(s) or decreased activity of
phosphatase(s) (Buee et al. 2000). Interestingly, some mutations of the amyloid precursor protein found in an early onset
AD lead to a marked increase in synthesis of N-terminally
truncated Ab peptides ending at the position of 42, whereas
Abx-40 is obviously not affected (Ancolio et al. 1999).
We did not find correlations between the concentrations of
Ab peptides in the CSF and blood. This might point to the
fact that the two sets of amyloid b peptides (in the CSF and
blood, respectively) come from different metabolic mechanisms, like e.g. release from independent populations of cells
in addition to diffusion through the blood-CSF barrier. To
clarify this point, certainly further studies are necessary.
Concluding, the analysis of the CSF concentration of
amyloid b species ending at the amino acid position of 42
might lead to misinterpretation of NDD outcome in subjects
with constitutively high- or low concentration of total Ab
peptides. We postulate that the amyloid b concentration ratio
(Abx-42/x-40) can possibly improve reliability of the
neurochemical dementia diagnosis.
Acknowledgement
This study has been supported by the following grants from German
Federal Ministry of Education and Research (BMBF): Kompetenznetz Demenzen (01 GI 0420), and HBPP-NGFN2 (01 GR 0447).
We gratefully appreciate technical help of Mrs. Christine Schödel
and Mrs. Ute Schulz.
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Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2006.04404.x