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Cerebrospinal fluid inhibits Alzheimer -amyloid fibril formation in vitro.

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abnormal gyrus on MRI, suggesting that the area of
ictal onset was localized to the abnormal region visualized by both MRI and SPECT. The images obtained
in Patient 2 had similar characteristics. These observations suggest that ictal SPECT may demonstrate prim a epileptogenesis
in cortical dysplasia.
Ictal SPECT must be performed at the onset of the
ictal episodes; delayed injections may demonstrate activation of secondary epileptogenic tissue and may lead
to erroneous conclusions. Although nonspecific for the
underlying pathology, ictal S P E C is extremely useful
in selected patients, as demonstrated here.
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Cerebrospinal Fluid Inhibits
Alzheimer p-Amyloid Fibril
Formation In Vitro
Thomas Wisniewski, MD,” Eduardo Castano, MD,?
Jorge Ghiso, PhD,t and Blas Frangione, MD, PhDT
Alzheimer’s disease is characterized by the deposition of
@-protein(A@)as amyloid. Recently, it was found that
AB is a normal component of serum and cerebrospinal
fluid. Synthetic peptides homologous to AP form amyloid-like fibrils spontaneously in water or physiological
solutions. Using a peptide homologous to AP1-40, we
find that fibril formation is inhibited by the presence of
cerebrospinal fluid.
Wisniewski T, Castano E, Ghiso J, Frangione B.
Cerebrospinal fluid inhibits Alzheimer P-amyloid
fibril formation in vitro.
Ann Neurol 1993;34:631-633
The central pathological event in Alzheimer’s disease
(AD) is the deposition of P-protein (A@)as amyloid
fibrils within senile plaques and cerebral blood vessels.
Until recently AP was thought to arise only by abnormal degradation of a larger precursor protein (PPP). It
is now known that AP is a normal component of serum, cerebrospinal fluid (CSF), and cell culture supernatants, present in 1 to 3 ngiml concentrations [ I , 2).
The initial sequence of soluble AP (SAP) revealed it
to consist of 40 amino acids [l, 21, which is homologous to cerebrovascular amyloid. However, it is likely
that the sequence of SAP will be as heterogeneous as
Aj3 deposited in amyloid.
Extensive prior studies have shown that synthetic
peptides homologous to Apl-40 and other AP peptides spontaneously form amyloid-like fibrils [3, 41.
Clearly the question arises of why SAP does not form
fibrils normally. The concentration will be an important
factor; however, it is also possible that specific inhibitors to fibril formation exist in physiological fluids. To
test this hypothesis we have used synthetic peptides
homologous to Apl-40 and studied the influence of
CSF on amyloid fibril formation by electron micros-
From the Departments of tPathology and *Neurology, New York
University Medical Center, New York, NY.
Received Mar 12, 1993, and in revised form May 27. Accepced for
publication May 27, 1993.
Address correspondence to Dr Wisniewski, Department of Neurology, New York University Medical Center, 550 First Avenue, TH
427, New York, NY 10016.
Copyright 0 1993 by the American Neurological Association 631
Fig 1 . Electron micrographs of structures formed by APl-40
before 31% reduction: bar represents 1 pm). (A)
Uranyl acetate negatively stained fibrils of peptides incubated
f i r 48 hours in phosphate-bufered saline. IB) API-40 peptides
incubated for 48 hours in cerebrospinal fEuid.
copy and a spectrofluorometric assay that utilizes the
novel fluorescence of amyloid fibrils in the presence
of thioflavine T [ S ] .
Methods and Results
Synthetic peptide DAEFRHDSGYEVHHQIUVFFAEDVGSNKGAIIGLMVGGVV (Apl-40, positions 597-636 of
APP,,,) was synthesized at the Center for the Analysis and
Synthesis of Macromolecule (State University of New York,
Stony Brook, NY) by solid-phase techniques. Crude peptide
was dissolved in 0.1% trifluoroacetic acid (TFA)/30% acetonitrile and purified via high performance liquid chromatography (HPLC) using a 20-prn Aquapore C8 (250 x 10 mm)
column (Brownlee) and a linear gradient of 30 to100% acetonitrile in 0.1% TFA at a flow rate of 2 ml/min. The column
effluent was monitored by absorbance at 214 nm. Solutions
of peptide were quantified by amino acid analysis using a
Pico-Tag analysis system (Waters) and the standard protocol.
The Apl-40 was dissolved in water at 1 mg/ml and mixed
1 : 1 with separate CSF samples of 5 normal and 5 A D patients (clinically determined). Controls were mixed with 2 x
phosphate buffer saline (PBS) (1 x = 20 mM phosphate,
150 mM NaCI, pH 7.4), 2 x PBS with human albumin (40
mg/dl), or a 2 x solution of ultrafiltrate of CSF. The latter
was prepared by ultrafiltrating CSF through a Omegacell (Filtron) with a l-kd cutoff. The solution was protein free by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). It was concentrated to 2 x by speed-vacuum
centrifugation. Peptide mixtures were incubated for 48 hours
ar room temperature and suspensions were prepared for negative staining using 2% uranyl acetate with a standard protocol [6]. In PBS, PBS-albumin, and CSF ultrafiltrate, numerous myloid-like fibrils were present ( F g 1A). These fibrils
were approximately 10 nm in diameter and up to 2 p,m in
length. In CSP both from AD and normal patients only occasional shorter fibrils were noted (Fig 1B).
632 Annals of Neurology Vol 34 No 4 October 1993
For the specrrofluorometric assay 20 pg by amino acid
analysis of Apl-40, from a stock solution in 0.1% TFA/
50% acetonitrile, were lyophilized and resuspended in 20
p1 of either PBS, pooled normal CSF, or pooled AD CSF.
Individual samples were incubated at room temperature for
10, 20, or 70 hours. At these times, samples in triplicate
were prepared for reading on a Hitachi 2000 spectrofluorometer, with thioflavine T (Sigma), using a previously described protocol [51. Excitation and emission wavelengths of
435 and 490 nrn respectively were used. Samples were read
every 10 seconds over 5 minutes and the readings were averaged (Fig 2). There was a greater than 50% inhibition of
fluorescence with Apl-40 in CSF compared with Apl-40
on PBS. Incubated CSF had no appreciable fluorescence.
There was no difference, under the conditions tested, between pooled AD and normal CSFs.
Prior studies using synthetic peptides have shown that
amyloid-like fibril formation is influenced by concentration, p H [3,4}, nucleation [5}, and mutations 16-91.
The presence of the familial AD, Dutch-type mutation
[lo} at residue 22 of AP, under different experimental
conditions, accelerates the rate of fibril formation [b],
increases aggregation 171, or increases fibril stability
[S}. Transfected cells expressing the Swedish familial
AD mutations (codons 595 and 596 of APP,,,) [ l l ]
produce higher quantities of SAP 112). It is likely that,
in addition, other factors such as altered binding of
SAP to carrier and/or charperone proteins may influence fibril formation. Our data show that CSF contains
one or more inhibitors of fibril formation, which we
designate as “desaggrin(s).” Recently it has been shown
that apolipoprotein E (apo E) 113, 141 and apolipoprotein J (apo J) [ l S } bind to SAP. Furthermore the isotype apo E4 is linked to sporadic and late onset familial
AD f141. These lipoproteins are abundant in both serum and CSF, normally functioning as transporters of
hydrophobic compounds such as lipids and fatty acids
AB1-40, Thiof lavin Fluorescence
Hours o f Incubation
Fig 2. Plot of tbefluore.icence (in arbitraq units) over hours
of incubation ofAB1-40 peptidc either in (a)pho.fphatebufjered saline or pooled normal cerebrospinal fluid 10).The
platted vulues represent the mean of t w o independent experiments. At each time point Jamples were run i n triplicate.
C13-151. We propose that they also bind and transport
hydrophobic peptides such as SAP. The interaction of
these proteins and SAP may be critical for amyloid
formation, as has already been suggested by Ap. { l b ] .
This research was supported by NIH grants KO8AG00542-01,
AR02594, and Lead Award AG10953, and grant IIRG-91-102 from
the Alzheimer’s Disease and Related Disorders Association.
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Brief Communication: Wisniewski et al: CSF Inhibition
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