From the Department of Molecular Animal Physiology, University of Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands
Received for publication, December 7, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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In patients with Alzheimer's disease or
Down's syndrome, the cerebellar cortex exhibits protein deposits in
neurofibrillary tangles and neuritic plaques. Recently, the
deposits have been shown to contain protein fragments of ubiquitin-B
and amyloid precursor protein (APP) with an aberrant carboxyl terminus
resulting from frameshift mutations (dinucleotide deletions; Alzheimer's disease
(AD)1 is a neurodegenerative
disorder of the brain and accounts for the most frequent form of
dementia at higher age. The disease is characterized by the presence of neurofibrillary tangles, neuropil threads, and neuritic plaques in the
brain. A major constituent of the plaques is To examine how widespread the phenomenon of molecular misreading is and
whether hot spots other than GAGAG may occur, we have developed a
bacterial expression system with the green fluorescent protein (GFP) as
a reporter and successfully used this system for the screening of
frameshift mutations in mRNAs even when the mutational rate is low.
We discovered novel frameshift mutations at different sites in Ubi-B
and APP gene transcripts isolated from cortical regions of aged individuals.
Isolation of RNA--
Using a standard Trizol procedure (Life
Technologies, Inc.), total RNA was extracted from cortical regions of
aged individuals that had suffered from AD or DS (patients 92-080 (DS,
58, RT-PCR--
Single stranded cDNA was synthesized by using 2 µg of total RNA, randomly primed with hexamers and reverse
transcribed with RT Superscript II (Life Technologies, Inc.). The PCR
was performed with PWO DNA polymerase (proofreading activity;
Roche Molecular Biochemicals). Initially, we experienced a technical
problem in that a substantial percentage (~1%) of the synthesized
primers was found to contain a deletion or an insertion of a
nucleotide. Therefore, primers (Biolegio, Malden, The
Netherlands) were purified by SDS-PAGE to diminish background signals
caused by the mistakes made during primer synthesis. The primers used
for Ubi-B mRNA analysis were
5'-ggggggggaagcttccgctatcaggtcaaaatg-3' as forward and
5'-gggggtctagatcttcacgaagatctgcat-3' as reverse primer (255-bp PCR
product). For APP mRNA analysis, the following primer sets were
used: APP-ex9 with 5'-gggggaagctttgcccatttccagaaagcc-3' as forward and
5'-gggggtctagacggcggtcattgagcatggc-3' as reverse primer (242-bp
product); APP-BA with 5'-ggggaagctttgggttgacaaatatcaagagc-3' as
forward and 5'-ggggtctagattctgcatctgctcaaagaac-3' as reverse primer
(APP-BA1; 341-bp product) or 5'-ggggtctagattttcgtagccgttctgctgc-3' as
reverse primer (APP-BA2; 312-bp product); APP-CT with
5'-gggggaagcttgaagaagaaacagtacaca-3' as forward and
5'-ggggtctagattctgcatctgctcaaagaac-3' as reverse primer (APP-CT1; 141 bp product) or 5'-ggggtctagattttcgtagccgttctgctgc-3' as reverse primer
(APP-CT2; 112 bp product).
Screening Strategy--
PCR products were gel-purified, digested
with HindIII and XbaI, and subcloned into the
bacterial vector pGFPuv (6079-1; CLONTECH; GenBankTM accession number U62636; see Fig. 1). DNA
was isolated from selected clones (Flexiprep kit, Amersham Pharmacia
Biotech), and insert sizes were determined. During the development of
this method we experienced a background signal caused by internal
initiation of translation at the first AUG of the GFP coding region. To
prevent this background signal, this AUG was removed via PCR with 5'
primer ggtcgactctagaaaaaagtaaaggagaagaacttttcactgg and 3' primer
ctcagttggaattcattatttgtagagctcatgcatgccatg. The mutated construct is
referred to as pGFP*uv and was used for the cloning of the PCR
products to identify the frameshift mutations. DNA sequence analysis
was performed according to the manufacturer's protocol (ABI Prism 310;
PerkinElmer Life Sciences). The screening strategy provides different
ways to detect frameshift mutations. The Ubi-B primers were designed in
such a way that clones containing a wild type insert would show no
fluorescence, whereas clones containing an insert with a dinucleotide
deletion would generate weak fluorescence. By changing the 3' primer
one could also search for deletions of one nucleotide. For the
detection of mutations in APP gene transcripts, the primers were
designed in such a way that the wild type APP is in frame with GFP,
whereas a frameshift would lead to nonfluorescent bacteria. However,
one of the wild type APP PCR fragments (generated with primers APP-BA1
and APP-BA2) was found to result in a nonfluorescent GFP fusion protein
because of the size of the insert. Surprisingly, in this case a number of fluorescent bacteria were still detected, and sequencing of these
clones revealed the presence of APP frameshift mutations. These
frameshifts caused an early termination of translation, and it is
likely that reinitiation of translation occurred to generate a
fluorescent GFP fusion protein.
Age-related frameshift mutations in gene transcripts have been
shown to occur in Brattleboro rats (5) and in patients who had suffered
from AD or DS (2). These frameshift mutations are generated at the RNA
and not the DNA level, and this process has been referred to as
molecular misreading (6). The frameshifts were detected with an
immunoscreening procedure implying the need for a specific antibody for
each mutation to be investigated. To circumvent the necessity for the
generation of an antibody for the detection of any new mutation, we
developed a bacterial expression cloning strategy involving the use of
GFP as a reporter protein to screen PCR fragments up to 300 nucleotides
in length for the presence of frameshift mutations (see "Experimental
Procedures"). To examine the sensitivity of the method and to show
that the mutations are not generated as a result of a PCR artifact,
constructs containing either wild type Ubi-B or Ubi-B with the
dinucleotide deletion previously described (GU or
GA) in or adjacent to GAGAG motifs in their mRNAs, a process
referred to as molecular misreading. We have now used a bacterial
expression system with the green fluorescent protein as a reporter to
screen gene transcripts from aged controls, Alzheimer's disease, and Down's syndrome for molecular misreading. Novel frameshift mutations at a number of locations in the transcripts of the ubiquitin-B and APP
genes were discovered (
GA,
G,
GU,
GG,
CA,
AU,
A,
AA,
C,
U, and insertion of an A). Interestingly, most
mutations were in close proximity of short simple repeats (GAGAG,
GGUGGU, GAGACACACA, UCAUCAUCA, CAAACAAA, and GAAGAAGAA), demonstrating that the GAGAG motif does not constitute the only hot spot for transcriptional errors. Unlike the previously detected aberrant APP
fragments, some of the novel ones have the potential to generate the
neurotoxic peptide
-amyloid. We conclude that during aging molecular
misreading is a widespread phenomenon.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-amyloid, a neurotoxic
peptide generated by processing of the type I transmembrane
-amyloid
precursor protein APP (1). Two different types of AD can be
discriminated, namely the early-onset (familial) and the late-onset
(sporadic) forms. The sporadic form accounts for more than 60% of all
AD cases, but the cause of the development of these cases is unknown. A
recent study provided a new view on the mechanism that may
underly aging and that in particular may contribute to the development
of the sporadic forms of AD and Down's syndrome (DS; see Refs. 2 and
3). The new view is referred to as molecular misreading and is based on
the finding of a novel type of dinucleotide deletion in or adjacent to
a GAGAG motif in the mRNA transcripts of two neuronal genes
encoding APP and the cytoplasmic garbage protein ubiquitin-B (Ubi-B)
(2). These mutations were found at the RNA and not the DNA level and resulted in frameshifts and proteins with an aberrant carboxyl terminus
(2-4). The same type of frameshift mutations may underlie other
neuropathologies and age-related diseases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
) and 93-048 (DS, 67,
) were obtained from the Netherlands
Brain Bank, Amsterdam, The Netherlands; patients 92-50-529 (control,
96,
), 92-50-692 (control with plaques, 85,
), 94-52-312 (control, 85,
), 95-50-665 (control, 84,
), 88-50-322 (AD, 85,
), 88-52-120 (AD, 83,
), 92-51-371 (AD, 81,
), 95-51-242 (AD,
79,
), and 95-51-014 (AD, 81,
) were obtained from the Department
of Pathology, University of Nijmegen, The Netherlands).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
GU; Ubi-B+1;
see Ref. 2) were used in different ratios ranging from 1:1 to 1:10000
(wild type:
GU). Even at a ratio of 1:10000 the (weakly) fluorescent
clones containing the frameshift mutation could still be detected at
the expected frequency. Subsequently, the validity of the procedure was
checked by searching for a frameshift that is known to occur in Ubi-B
gene transcripts isolated from human DS brain tissue (2). A GU deletion
following a GAGAG motif in the Ubi-B transcript identical to the one
reported earlier (2) was indeed found (Table
I). Because the PCR primers corresponded to regions located in separate exons of the Ubi-B gene, the size of the
PCR fragment (255 bp) revealed that the mutations were detected at the
RNA and not the DNA level. Interestingly, in addition to the
GU, we
discovered several novel mutations (
GA,
G,
GG, and insertion
of an A) (Table I). These novel mutations were also located adjacent to
the GAGAG motif described earlier, except for the insertion of an A,
which occurred adjacent to a GGUGGU motif. This latter observation
indicates the importance of simple repeats for the process of molecular
misreading.
Frameshift mutations detected in human brain RNA of aged individuals
(controls (C) and patients that had suffered from AD or DS)
Another transcript previously shown to contain dinucleotide
deletions at a GAGAG motif is produced by the APP gene (2). In exons 9 and 10 of the APP gene, three GAGAG motifs are clustered. When applying
our strategy on RNA derived from aged individuals, we were again able
to confirm the previously observed GA deletion in the second GAGAG
motif in exon 9 (Table I). Furthermore, we detected a CA deletion in
exon 11 that, remarkably, occurred in a GAGACACACA motif, again
pointing to the involvement of simple repeats in molecular misreading.
Molecular misreading in neither exon 9 nor exon 11 of the APP gene can,
however, by itself lead to an increase in the production of A,
because the APP fragments generated by these frameshift mutations do
not contain the A
peptide region (encoded by exons 16 and 17 of the
APP gene). In contrast, the occurrence of a frameshift mutation in a
region of the mRNA corresponding to the C-terminal cytoplasmic
region of APP would lead to an aberrant protein with the potential to give rise to A
(Fig. 1). We therefore
searched for mutations in the mRNA region encoding the C-terminal
part of APP, and this analysis revealed a number of novel frameshift
mutations, namely
A and
AA (both in
UCAUCAUCAAAAAUUGG),
A
(UUCAAACAAAGGUGCAAUCAUUC),
G
(UUGGACUCAUGGUGGGCGGUUGUU;
UUCAAACAAAGGUGCAAUCAUUC; GAAGAAGAAACAGUACACA),
U
(UGAUCGUCAUCACACCU), and
C
(CTGTCACCCCAGAGGAG) (Table I). Some of
these mutations occurred in close proximity of short simple repeats
(UCAUCAUCA, CAAACAAA, and GAAGAAGAA). The mutations were not only
detected in mRNAs derived from patients who had suffered from AD or
DS but also in temporal cortex mRNAs of aged controls.
Interestingly, the control that showed the highest mutation frequency
exhibited plaques in the brain.
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The aberrant proteins resulting from molecular misreading might affect
cell physiology. For instance, the previously published Ubi-B+1 protein, lacking Glycine residue 76 that is
essential for recognition of the target protein (7), is no longer able
to ubiquitinilate proteins in
vitro.2 The
above-mentioned novel frameshift mutations in the Ubi-B gene transcripts probably also result in ubiquitin molecules unable to
function properly. Concerning the APP protein one should consider its
cytoplasmic tail that contains two sorting signals (YTSI and YENPTY),
characterized as internalization and endosomal/lysosomal targeting
signals, and necessary for intracellular trafficking of APP. Deleting
or mutating these signals indeed affects APP routing (8-13). The
frameshift deletions G at nucleotide 2103 and
C at nucleotide
2153, in the part of the APP transcript encoding the cytoplasmic
region, result in proteins with an aberrant and elongated cytoplasmic
tail lacking one or both of the targeting signals of APP (Fig. 1).
Because an affected APP routing may lead to an increased production of
A
(14), the novel APP proteins with the aberrant tails may give rise
to this neurotoxic peptide.
Although the frameshift mutations were found at a relatively low rate
(1:950 to 1:8600 transcripts; see Table I), at the single cell level
the frequency may be considerably higher. Immunocytochemical analysis
has revealed that only a small percentage of cells contain the aberrant
frameshift proteins, indicating that molecular misreading is indeed
restricted to a minority of neuronal cells (2). In an affected cell,
the percentage of mutated transcripts may well be relatively high,
which could significantly contribute to the malfunctioning of such a
cell, possibly eventually even promoting its degeneration. We conclude
that during aging molecular misreading is a widespread event occurring
at a variety of short simple repeats.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the Netherlands Institute for Brain Research (Amsterdam, The Netherlands) and the Department of Pathology (University of Nijmegen, The Netherlands) for providing human brain material. Furthermore, we thank J. Eygensteyn for technical assistance and Dr. F. van Kuppenveld, R. P. M. Vloet, Dr. F. W. van Leeuwen, and Dr. N. H. Lubsen for contributions.
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FOOTNOTES |
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* This research was supported by Grant 7F99.(2).47 from the Hersenstichting Nederland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 31-24-3610564;
Fax: 31-24-3615317; E-mail: gmart@sci.kun.nl.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M011040200
2 F. A. A. Weijts, W. H. van den Hurk, and G. J. M. Martens, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: AD, Alzheimer's disease; APP, amyloid precursor protein; DS, Down's syndrome; Ubi-B, ubiquitin-B; GFP, green fluorescent protein; PCR, polymerase chain reaction; bp, base pair.
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