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Correspondence |
1University of California-San Diego La Jolla, California;
2Yokohama City University School of Medicine Yokohama,
Japan
To the Editor-in-Chief:
In 1993, Saitoh and colleagues identified the non-Aß component
of Alzheimer’s disease
(AD), amyloid
(NAC), as an important
element of amyloid-enriched fractions in AD brains.1
NAC
is a 35-amino acid (aa)
fragment derived from its 140-aa precursor, 
-synuclein, which is now
recognized to play a major role in Lewy body disease
(LBD)
pathogenesis.2
Furthermore, immunohistochemical studies
with the anti-NAC-X1 antibody revealed that NAC is closely colocalized
with Aß in the AD plaques.1
Moreover, biochemical
studies showed that NAC is extremely hydrophobic and easily forms
amyloid-like fibrils under physiological conditions.3
These results led us to the hypothesis that NAC might play an important
role in Aß aggregation and amyloidogenesis in AD.1
In a recent issue of The American Journal of Pathology, Culvenor et al4 reported that NAC may not be associated with Aß in the plaques. Using the antibody NAC42580 to label cortical and hippocampal sections of AD cases, the authors found no evidence of NAC immunoreactivity in the plaques. Furthermore, although the NAC42580 showed certain immunoreactivity in the urea extracts of the sodium dodecyl sulfate (SDS)-insoluble fractions in diffuse LBD and Parkinson’s disease (PD) cases, there was no correlation between NAC and Aß immunoreactivity in the same AD fractions. Based on this study and on a previous report,5 the authors concluded that NAC may not be associated with Aß in the plaques of AD.
Several possibilities could be considered to explain the discrepancy
between our results and those reported by Culvenor et al.4
First, differences in immunoreactivity could be due to differential
properties of antibodies used in each study. NAC-X1 and NAC42580 were
raised against epitopes derived from different portions of the NAC
region: NAC1–9 for NAC-X11
and NAC15–31 for
NAC42580.4,5
It is important to note that immunoblotting
analysis showed that NAC-X1 preferentially recognizes aggregated forms
of the NAC molecule over the monomeric ones.3
Furthermore,
in our experience, NAC-X1 does not immunoreact with soluble
-synuclein.3
Thus, it can be predicted that our NAC-X1
antibody may be ideal to detect aggregated NAC in the plaques of AD. In
contrast, immunoblotting analysis with the NAC42580 showed
immunoreactivity with soluble -synuclein in brain
tissues.4,5
In fact, biochemical analysis of the NAC
peptide by El-Agnaf et al6
showed that the N-terminal
region of NAC (NAC1–18)
aggregates to form amyloid fibrils, while the C-terminal region
(NAC19–35) remains
soluble, suggesting that the N-terminal portion is essential for
aggregation of NAC peptide.6
Taken together, these
observations might suggest that the NAC1–9 peptide could be naturally
aggregated during inoculation in the rabbit, leading to the production
of NAC-X1, which preferentially recognizes aggregated forms of
NAC, whereas the C-terminal region of NAC may be less effective.
Second, differences in antibody immunoreactivity might depend on the methods used for tissue preparation. In this regard, the NAC-X1 antibody immunolabeled plaques in paraformaldehyde-fixed vibratome sections, but not in formalin-fixed, paraffin-embedded tissue.1,3,7,8 In fact, both Bayer and Culvenor used archival formalin-fixed and/or paraffin-embedded tissues, rather than vibratome, for their immunohistochemical studies,4,5 raising the possibility that fixation, solvents, and paraffin might destroy the NAC-X1 epitope. Further supporting the importance of tissue pretreatment and processing to detect the NAC epitope, we have recently shown that in vibratome sections pretreated with formic acid, the NAC-X1 antibody immunostained not only amyloid plaques and amyloid angiopathy, but also astroglial cells and granular neurons in LBD.7
Finally, it is possible that the NAC-X1 antibody might be cross-reacting with or recognizing protein quaternary structure. In this regard, immunoblotting analysis confirmed that both monomeric and aggregated forms of Aß were not immunoreactive with NAC-X1.1 Therefore, it is unlikely that NAC-X1 cross-reacts with Aß.
In summary, we would like to argue that it is necessary to continue
exploring the hypothesis that NAC plays an important role in plaque
formation and AD. The essential difficulty in obtaining conclusive
evidence may be due to the lack of information as to the mechanisms by
which NAC is generated from its precursor, -synuclein.2
In this regard, the results of this study by Culvenor et
al4
are potentially interesting, because their
immunoblotting analysis of brain homogenates showed that in addition to
full length -synuclein at 18kd, NAC42850 recognized faint bands at
12 and 6 kd, supporting the possibility that -synuclein undergoes
degradation. We expect that further characterization of these short
fragments might disclose a yet unknown mechanism of -synuclein
proteolysis, leading to a better understanding of the role of
-synuclein and NAC in the pathogenesis of AD.
References
-synuclein are not associated with Aß amyloid. Am J Pathol 1999, 155:1173–1181
-synuclein accumulates in Lewy bodies in Parkinson’s disease and dementia with Lewy bodies but not in Alzheimer’s disease ß-amyloid plaque cores. Neurosci Lett 1999, 266:213-216[Medline]
-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol 1999, in press
-synuclein distribution in Lewy body disease. Lab Invest 1998, 78:1169-1177[Medline]
1University of Heidelberg Heidelberg, Germany;
2University of Melbourne Parkville, Australia
Author’s Reply:
The central questions we have tried to address are whether intact
-synuclein (SN) or
its proteolytic product, the non-Aß component of Alzheimer’s disease
(AD) amyloid, NAC,
SN(61–95), ever
occurs as an extracellular aggregate, and, if so, whether there is any
special relationship with Aß deposition. Earlier studies by Saitoh,
Masliah, and colleagues1–4
using antibodies to
NAC(1–9) indicated that
it may be an integral but minor component of Aß amyloid plaques. Our
antibodies to
NAC(15–31)5,6
and NAC(11–23)
(unpublished) fail to
reveal an association of NAC with the extracellular Aß amyloid
deposits in AD. There is however, intracellular NAC immunoreactivity in
abnormal neuritic structures in close association with Aß amyloid
plaques restricted to the CA1 region of the hippocampus in cases of AD
combined with cortical Lewy bodies.
The response of Hashimoto and colleagues suggests our data reflect a
lack of reactivity with aggregated NAC; however, all our antibodies
react with aggregated SN as found in Lewy bodies. The suggestion
that differences may be due to tissue preparation protocols is also
unlikely, because our antibodies were reactive with Lewy bodies after
formalin fixation, paraffin sectioning, and formic acid treatment.
Hashimoto and colleagues themselves report no difference of labeling of
vibratome or paraffin sections after formic acid pretreatment of
tissue.4
We therefore suspect that the reactivity of the
NAC(1–9) antibody with
AD plaques may be due to cross-reactivity with Aß, since residues
6–8 of NAC share identity with the C-terminal region of
Aß(36–38), as
identified earlier by Han et al.7
Our Western blot analysis of SDS-insoluble extracts of brain tissue
from cases of dementia with Lewy bodies indicates that aggregates of
SN contain full-length as well as truncated species, including a
putative NAC fragment of about 6 kd. In pure AD cases, we are unable to
demonstrate accumulation of SDS-insoluble SN or a NAC fragment in
the same fractions that contain the SDS-insoluble Aß
peptide.5
In a further study of four AD cases,
SDS-insoluble SN or the NAC fragment was not detectable
(Campbell et al, manuscript
submitted). Our combined immunocytochemical and
biochemical data therefore indicate that SN and its proteolytic
products accumulate largely in intracellular compartments, independent
of extracellular Aß amyloid deposits.
References
-synuclein distribution in Lewy body disease. Lab Invest 1998, 78:1169-1177
-synuclein are not associated with Aß amyloid. Am J Pathol 1999, 155:1173–1181
-synuclein accumulates in Lewy bodies in Parkinson’s disease and dementia with Lewy bodies but not in Alzheimer’s disease ß-amyloid plaque cores. Neurosci Lett 1999, 266:213-216
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