(Received for publication, July 24, 1995)
From the
Abnormal expression of human amyloid precursor protein (hAPP) gene products may play a critical role in Alzheimer's disease (AD). Recently, a transgenic model was established in which platelet-derived growth factor (PDGF) promoter-driven neuronal expression of an alternatively spliced hAPP minigene resulted in prominent AD-type neuropathology (Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T., Gillespie, F., Guido, T., Hagopian, S., Johnson-Wood, K., Khan, K., Lee, M., Leibowitz, P., Lieberburg, I., Little, S., Masliah, E., McConlogue, L., Montoya-Zavala, M., Mucke, L., Paganini, L., and Penniman, E.(1995) Nature 373, 523-527). Here we [Medline] compared the levels and alternative splicing of APP transcripts in brain tissue of hAPP transgenic and nontransgenic mice and of humans with and without AD. PDGF-hAPP mice showed severalfold higher levels of total APP mRNA than did nontransgenic mice or humans, whereas their endogenous mouse APP mRNA levels were decreased. This resulted in a high ratio of mRNAs encoding mutated hAPP versus wild-type mouse APP. Modifications of hAPP introns 6, 7, and 8 in the PDGF-hAPP construct resulted in a prominent change in alternative splice site selection with transcripts encoding hAPP770 or hAPP751 being expressed at substantially higher levels than hAPP695 mRNA. Frontal cortex of humans with AD showed a subtle increase in the relative abundance of hAPP751 mRNA compared with normal controls. These data identify specific intron sequences that may contribute to the normal neuron-specific alternative splicing of APP pre-mRNA in vivo and support a causal role of hAPP gene products in the development of AD-type brain alterations.
A number of observations indicate that the disregulated
expression or processing of human amyloid protein precursor
(hAPP) (
)gene products may play a critical role in the
development of Alzheimer's disease (AD). Increased cerebral
deposition of the hAPP-derived peptide A
in the form of amyloid
plaques constitutes a hallmark of AD(2) . Persons with
Down's syndrome who carry an additional copy of the hAPP gene on
their third chromosome 21 show an overexpression of hAPP (3, 4) as well as a prominent tendency to develop
AD-type pathology early in life(5) . A number of hAPP point
mutations that are tightly associated with the development of familial
AD encode amino acid changes close to either side of the A
peptide
(for review, see (6) and (7) ), and in vitro studies indicate that aggregated A
can induce
neurodegeneration (for review, see (8) ).
To assess the
neuropathogenic potential of hAPP gene products in vivo, a
variety of transgenic models have been developed that were engineered
to overexpress either full-length hAPP or hAPP derivatives in the CNS
using different
promoters(1, 9, 10, 11, 12) .
So far, only one transgenic model has reproducibly developed prominent
AD-type neuropathology: neuronal expression of an alternatively spliced
minigene, driven by the platelet-derived growth factor (PDGF B chain)
promoter and encoding (Val
Phe)-mutated hAPP770,
hAPP751, and hAPP695, resulted in an age- and brain region-dependent
development of typical amyloid plaques, dystrophic neurites, loss of
presynaptic terminals, astrocytosis, and microgliosis(1) . In
contrast, previous transgenic models with neuron-specific enolase (NSE)
promoter-driven expression of individual hAPP isoforms, such as
wild-type or (Val
Ile)-mutated hAPP695 or hAPP751,
showed no (12) or only minimal (13) pathology.
The APP gene contains 19 exons (Fig. 1A), three of which (exons 7, 8, and 15) are subject to alternative splicing. The different APP isoforms that are derived from this gene are designated according to their number of amino acids. The longest isoform, APP770, contains a 56-amino acid domain (encoded by exon 7), which shares sequence homology with, and can function like Kunitz-type serine protease inhibitors (KPI) as well as an adjacent 19-amino acid domain (encoded by exon 8) with homology to the MRC OX-2 antigen, which is found on the surface of neurons and certain immune cells (for review, see Refs. 12, 14, and 15). APP695 lacks both of these domains (Fig. 1A) and is produced primarily by neurons, which constitute the primary source of APP within the CNS(16, 17) . Brain tissue expresses little APP770 and, depending on the animal species and brain region analyzed, low, intermediate, or high levels of APP751(16, 18, 19, 20, 21) . The latter isoform contains the KPI domain (exon 7) but lacks the OX-2 domain (exon 8) (Fig. 1A). In culture, astrocytes, and microglia express predominantly KPI-encoding APP mRNAs(22, 23) ; their expression of APP and preferred APP splicing pattern in vivo are less well established. Human white matter astrocytes have been shown to express APP mRNA by double labeling with antisense APP cRNA probes and antibodies against glial fibrillary acidic protein(15) . Although acute excitotoxic brain injury induced increased expression of APP (24) and KPI(25) , immunoreactivities in reactive astrocytes of the rat hippocampus, reactive astrocytes in the hippocampi of AD brains with chronic gliosis showed no clear overexpression of APP mRNAs(17, 26) . For a comprehensive quantitation of different APP splice products in the CNS and peripheral organs of normal rodents see (27) .
Figure 1: Diagrammatic comparison of the hAPP gene and hAPP-encoding transgenes. A, exon-intron organization of the hAPP gene based on (34) . Boxes represent exons, closed and hatched boxes represent isoform-specific exons as shown. Horizontal lines indicate introns. The whole gene is approximately 400 kb in length. Elements are not drawn to scale. B, fusion gene constructs used to express hAPPs in neurons of transgenic mice. The regulatory sequences incorporated into the transgenes were derived from either the rat NSE gene or the human PDGF B chain gene as described in Refs. 12 and 1, respectively. Numbered boxes represent hAPP exons. Elements are not drawn to scale. The NSE-driven hAPP cDNAs were either wild-type or point mutated to encode the familial AD-associated valine to isoleucine substitution(85) . The PDGF-driven hAPP minigene was point mutated to encode a valine to phenylalanine change at the same position; this change is also tightly linked with familial AD(86) . Besides the introduction of a point mutation into exon 17, hAPP coding sequences were not modified. Note, however, that the three introns incorporated into the PDGF-hAPP construct were modified as described under ``Materials and Methods'' and panel C below. As indicated, alternative splicing of pre-mRNA derived from the PDGF-hAPP transgene results in transcripts encoding hAPP770, hAPP751, or hAPP695. C, intron modifications of the PDGF-hAPP transgene. Compared with the authentic hAPP gene (top), all three introns of the PDGF-hAPP transgene (bottom) were modified. B, authentic BamHI sites. a and b, engineered BamHI sites introduced via PCR primer modifications. X, XhoI site; R, EcoRI site. Crossed out line, deleted intron sequence. Elements are not drawn to scale. Numbers indicate distances in base pairs; *, based on (34) ; , based on PCR/sequence analysis. For the construction of PDGF-hAPP intron 6, genomic hAPP sequence extending from 143 bp downstream of exon 6 (site a) to the BamHI site 1,658 bp upstream of exon 7 was deleted. No sequences were deleted from intron 7; however, the single XhoI site in hAPP intron 7 was eliminated by XhoI digestion, filling in of ends with Klenow and religation (X); this modification adds 4 bp to intron 7. For the construction of PDGF-hAPP intron 8, genomic hAPP sequence extending from the first BamHI site at 2,329 bp downstream of exon 8 to 263 bp upstream of exon 9 (site b) was deleted. Note that this map differs from a previously published report on the structure of the hAPP gene (34) (see text for interpretation). D, size estimation of hAPP intron 7 using PCR analysis of genomic DNA. Genomic DNA samples extracted from HeLa cells (lane 1) or from tail tissues of a PDGF-hAPP mouse (lane 2) and a nontransgenic mouse (lane 3) and a control sample containing no DNA (lane 4) were amplified as described under ``Materials and Methods'' using a hAPP exon 7 upstream primer (5`-GAGGTGTGCTCTGAACAAGC-3`) and a hAPP exon 8 downstream primer (5`-ATCTCGGGCAAGAGGTTC-3`). Amplification products were separated electrophoretically on a 0.6% agarose gel and stained with ethidium bromide. The gel photograph was contrast-inverted. L, ladder of size standards. Note the similar size of the amplification products obtained from human genomic DNA (lane 1) and from genomic murine DNA containing multiple copies of the PDGF-hAPP transgene (lane 2).
In the current study, we compared the levels and alternative splicing of transcripts encoding APP695, APP751, and APP770 in brain tissue of PDGF-hAPP and NSE-hAPP mice and of humans with and without AD to address the following questions in vivo. (i) Could the development of prominent AD-type pathology in PDGF-hAPP mice be related to the level of total APP expression achieved in this model or to the relative proportion of mutated hAPP versus wild-type mouse APP (mAPP) expressed in their brains? (ii) Does overexpression of hAPP in the murine CNS alter the expression of the endogenous mAPP gene? (iii) How is the neuron-specific splicing of hAPP exons 7 and 8 affected by specific modifications of hAPP intron sequences made in the construction of the PDGF-hAPP transgene? (iv) Does the expression of hAPP transcripts in brains of hAPP transgenic models resemble the expression of hAPP transcripts found in the CNS of humans with AD?
Figure 2: Regions of endogenous and transgene-derived APP mRNAs protected by antisense riboprobes. A: hAPPSV40, mAPP, and total APP probes. The 3`-ends (coding region, 3`-UTR, poly(A) signal, and tail) of NSE-hAPP (top), PDGF-hAPP (middle) and endogenous murine APP (bottom) mRNAs are depicted diagrammatically. The hAPP 3`-UTR of NSE-hAPP constructs was truncated at the HindIII site (nucleotide 3023 in Genbank accession number X06989), whereas the hAPP 3`-UTR of the PDGF-hAPP construct ends 135 bp downstream of the first hAPP poly(A) signal(87) . In RPAs, only the regions that hybridize with complete or (depending on the conditions used) near-complete complementarity with specific riboprobes will be protected (black, stippled, or hatched), whereas noncomplementary regions will be degraded (open). Numbers indicate the sizes of protected fragments in nucleotides. The hAPPSV40 probe was produced by ligating hAPP with SV40 sequences via a NotI linker. This probe protects an hAPP fragment that is present in all endogenous hAPP mRNAs as well as in the hAPP transgenes; because of significant sequence variation in the 3`-UTR of these transcripts, this probe does not protect mAPP or APLP2 mRNAs under the conditions used here. The hAPPSV40 probe also protects a smaller fragment of SV40 sequence, which is present in hAPP transgenes but not in endogenous human or murine transcripts. Because the NotI linker is absent from hAPP transgenes, hybrids between transgene-derived mRNAs and the hAPPSV40 probe are cleaved into two fragments (Fig. 3A). The mAPP probe specifically protects a region of mAPP 3`-UTR present in all mAPP transcripts; it does not protect hAPP or APLP2 mRNAs. The total APP probes protect a fragment of APP coding region in which hAPP and mAPP transcripts show a high degree of homology; under appropriate conditions, this probe protects both hAPP and mAPP mRNAs (see Fig. 4for further details). B, alignment of sequences protected by hAPP770 and mAPP770 riboprobes. The DNA template for the hAPP770 probe was amplified by PCR from a cloned hAPP770 cDNA (a generous gift from Dr. D. Goldgaber, SUNY, Stony Brook, NY). The mAPP770 probe template was generated by reverse transcriptase PCR from mouse brain RNA (see ``Materials and Methods''). Sequence analysis of two different mAPP770 clones, amplified from a Balb/c or a SJL/J mouse, respectively, gave the same results (shown above). This sequence is consistent with previous publications reporting on different components of this region(37, 38, 88) . Because there are numerous mismatches (*) between hAPP and mAPP sequences in this region, the hAPP770 probe generally protects only hAPP and the mAPP770 only mAPP transcripts. While neither of these probes would be expected to protect APLP2 mRNAs(36, 37, 38, 39, 40) , these probes cannot differentiate between APP transcripts that contain or lack exon 15; however, the latter (L-APP mRNAs) are expressed in brain at very low levels(27) . C, APP mRNA regions protected by hAPP770 (h) and mAPP770 (m) riboprobes. To quantitate the three main APP splice products (APP770, APP751, and APP695), two probes were generated from human or murine APP770 cDNAs, respectively, that extend from exon 6 into exon 9 (see panel B above). The expected sizes of the protected mRNA fragments for the different isoform-specific transcripts are indicated (nt, nucleotides). Solid lines, protected probe regions (bands on RPA autoradiograph). Dotted lines, unprotected probe regions (degraded by RNase treatment).
Figure 3:
hAPP mRNA levels in brains of transgenic
mice and of humans. Total RNA was extracted from hemibrains of
nontransgenic controls (Non-Tg) and of transgenic
mice from lines NSE-hAPP751m-57, NSE-hAPP695m-19, and PDGF-hAPP-109
(three mice/group) as well as from the frontal cortex of humans. Levels
of hAPP mRNA were quantitated by RPA using the hAPPSV40 probe (Fig. 2A) as described under ``Materials and
Methods.'' A, representative autoradiograph. Lane u shows signals of undigested radiolabeled probes (indicated on
left; tRNA but no RNase added). The other lanes contained the same
riboprobes plus brain RNA samples, digested with RNase (condition 2). Labels and arrows on right indicate the
expected size for specific protected mRNA fragments following digestion
of noncomplementary probe sequences (vector-derived) and transcripts.
Actin signals were used as a control for RNA content/loading. Numbered
lanes: 1, normal control; 2 and 3, cases
with moderate AD. B, PhosphorImager quantitation of the
signals shown in A above. Columns and error bars represent means and S.E., respectively. Similar results were
obtained in an additional experiment using
poly(A)-enriched RNA (0.5 µg/case) extracted from
an independent set of comparable samples (not shown). C,
autoradiograph comparing hAPP mRNA levels in human controls and cases
with different degrees of AD. mod., moderate; sev.,
severe. Conventions otherwise as in panel
A.
Figure 4:
Comparison of total APP mRNA levels in
brains of transgenic mice and of humans with AD. Total RNA was
extracted from hemibrains of nontransgenic controls (Non-Tg) and of transgenic mice from lines
NSE-hAPP751m-57, NSE-hAPP695m-19, and PDGF-hAPP-109 (three mice/group)
as well as from the frontal cortex of humans with moderate (mod.) AD (n = 3). Total APP mRNA levels were
determined by RPA using total APP probes that protect a region of high
homology between hAPP and mAPP transcripts (Fig. 2A).
RNase condition 1 was used to promote similar protection of hAPP and
mAPP transcripts. A, autoradiograph showing results obtained
using a total APP probe generated from a human cDNA template (total
APP probe). Conventions as in Fig. 3A. B, PhosphorImager quantitation of the signals shown in A above. C, PhosphorImager quantitation of signals obtained
on the same samples using a total APP probe generated from a murine
cDNA template (total APP
probe). See text for
interpretation of results.
Levels of specific mRNAs were determined using solution
hybridization RNase protection assays (RPA) as described previously (33) with the following modifications. Hybridization was done
in a 10-µl volume at 55 °C. RNase concentrations were optimized
for different probes and applications; the following conditions were
used routinely: condition 1 = 0.2 µg/ml RNase A + 600
units/ml RNase T; condition 2 = 2.0 µg/ml RNase
A + 1,500 units/ml RNase T
; condition 3 = 40
µg/ml RNase A + 80 units/ml RNase T
; condition 4
= 200 µg/ml RNase A + 1,500 units/ml RNase
T
. In each case, 100 µl of the RNase mix was added per
10-µl sample. Unless indicated otherwise, each sample contained 10
µg of total RNA plus 20 µg of tRNA. Samples were separated on
5% acrylamide, 8 M urea TBE gels, and dried gels were exposed
to Kodak XAR film (Eastman Kodak Co.).
Compared with nontransgenic controls, PDGF-hAPP mice showed a 4-6-fold increase in total APP mRNA levels (Fig. 4). In contrast, the highest NSE-hAPP expressor line showed on average only a 1.3-1.6-fold increase in total APP mRNA levels over levels found in nontransgenic controls. This increase is lower than the approximately 2.9-fold increase in m/hAPP mRNA expression determined for the same line of NSE-hAPP mice by Northern blot analysis in a previous study using a large hAPP751 cDNA probe(12) . It is likely that this cDNA probe showed substantially greater bias toward transgene-derived hAPP transcripts than the total APP riboprobes chosen for the above RPA analysis and, hence, overestimated the level of total APP mRNA overexpression in NSE-hAPP mice. The 1.3-1.6-fold increase in total APP mRNA levels determined here is clearly more consistent with the 1.33-1.52-fold increase in total APP protein expression determined for this line by PhosphorImager analysis of radiolabeled Western blots(12) .
Figure 5: Effect of hAPP overexpression on mAPP mRNA levels. Total RNA was extracted from hemibrains of nontransgenic controls (Non-Tg) and of transgenic mice from lines NSE-hAPP751m-57, NSE-hAPP695m-19, and PDGF-hAPP-109 (three mice/group). Levels of endogenous mAPP transcripts were measured by RPA using an mAPP-specific probe (Fig. 2A) and RNase condition 2. Results indicate means ± S.E. of radioactive signals as determined by PhosphorImager quantitations. Note the significant decrease in mAPP mRNA levels in the PDGF-hAPP mice (p value determined by Student's t test comparing PDGF-hAPP mice with nontransgenic controls). Similar results (not shown) were obtained in two additional experiments using independent sets of comparable samples and quantitations of signals generated with either the mAPP probe or the mAPP770 probe (86-nucleotide fragment of mAPP exon 9; see Fig. 2C and Fig. 6A).
Figure 6: Quantitation of mAPP and hAPP splice products. RNA was extracted from hemibrains of nontransgenic controls (Non-Tg) and of transgenic mice from lines NSE-hAPP751m-57, NSE-hAPP695m-19, and PDGF-hAPP-109 (three to four mice/group) (A and B) and from the frontal cortex of humans without (Controls; n = 5) or with AD (n = 6) (C). Ten µg of total RNA was analyzed per sample using RNase condition 2 (C) or 3 (A and B). mAPP (A) or hAPP (B and C) splice products (indicated on the right) were detected using the mAPP770 probe or the hAPP770 probe, respectively (see Fig. 2, B and C). Representative autoradiographs are shown. For a statistical analysis of the relative abundances of isoform-specific APP mRNAs, see Table 2. The mAPP (exon 9) band in panel A represents an 86-nucleotide fragment of mAPP exon 9 (Fig. 2C). To distinctly reveal the prominent hAPP770 and hAPP751 bands in PDGF-hAPP brain samples, the autoradiograph in panel B was developed after a 20-min exposure; because of this short exposure, the actin bands appear faint.
A comparison of endogenous mAPP splice products in brains of nontransgenic and PDGF-hAPP mice (n = 3/group) using the mAPP770 probe and RNase conditions 3 or 4 indicated that the significant drop in mAPP mRNA levels in PDGF-hAPP mice (Fig. 5) was due primarily to a prominent decrease in mAPP695 levels, whereas alterations in mAPP770 and mAPP751 mRNA levels were less pronounced (data not shown). Although the overall trends were clear, precise quantitations of these changes were difficult because degradation products of the abundant hAPP770 and hAPP751 mRNAs interfered with the reliable measurement of isoform-specific mAPP signals in the PDGF-hAPP mice.
The current study demonstrates that 2-4-month-old
PDGF-hAPP mice from a line that consistently develops prominent AD-type
pathology between 6 and 8 months postnatally express substantially
higher hAPP and total APP steady-state mRNA levels than NSE-hAPP mice
from lines that fail to develop such alterations, suggesting that high
levels of hAPP expression may be requisite for the induction of robust
pathology. PDGF-hAPP mice were analyzed before the development of
amyloid deposits to help differentiate potentially causal factors from
secondary responses that could follow amyloid-induced neuronal injury.
The high levels of hAPP expression in PDGF-hAPP mice were associated
with a statistically significant 30% reduction in mAPP mRNA levels (Fig. 5). Because levels of other neuronal messages encoding
MAP-2, GAP-43, synaptophysin, or NFL were not significantly altered in
PDGF-hAPP mice at this developmental stage, it is unlikely that the
decrease in mAPP mRNA levels resulted from a generalized decrease in
the expression of endogenous neuronal gene products. It is tempting to
speculate that the decrease in mAPP message levels reflects an active
counterregulatory decrease in the expression of the endogenous mAPP
gene in cells exposed to high levels of hAPP. The findings that total
APP levels in the highest NSE-hAPP expressor line were increased
1.3-1.6-fold over levels found in nontransgenic controls (Fig. 4), while endogenous mAPP levels were not significantly
altered (Fig. 5), indicates that in the brains of these mice
only about 23-38% of APP transcripts encode mutated hAPP. In
contrast, the 4-6-fold increase in total APP levels in the
PDGF-hAPP mice (Fig. 4), in combination with their 30% decrease
in endogenous mAPP levels (Fig. 5), indicates that in the brains
of these mice, approximately 83-88% of APP transcripts encode
mutated hAPP. In concert with the substantial increase in total APP
mRNA levels, this prominent shift toward mutated hAPP transcripts can
be expected to strongly promote the synthesis of amyloidogenic hAPP
molecules in the CNS of PDGF-hAPP mice. Consistent with this
prediction, significant increases in A production have been
detected in brains of PDGF-hAPP mice(1) . Experiments are in
progress to further complement the RNA analysis presented here with
characterizations of these models at the protein level.
It is interesting that PDGF-hAPP-derived pre-mRNA was spliced differently than endogenous hAPP pre-mRNA in humans or mAPP pre-mRNA in mice. In nontransgenic mice and normal human controls, APP695 transcripts were substantially more abundant than APP770 transcripts with APP751 mRNA levels being similar to APP770 mRNA levels in mice and closer to APP695 mRNA levels in humans ( Fig. 6and Table 2). In contrast, alternative splicing of PDGF-hAPP pre-mRNA resulted in roughly similar levels of hAPP770 and hAPP751 transcripts, both of which were expressed at high abundance, and in comparatively low levels of hAPP695 mRNA (Fig. 6B and Table 2). In fact, hAPP695 mRNA levels in PDGF-hAPP mice were similar to those achieved in our highest NSE-hAPP695 expressor line (Fig. 6B). This implies that the difference between these lines with respect to hAPP and total APP mRNA levels ( Fig. 3and 4) is due primarily to the substantial overexpression of KPI-containing isoforms achieved in the PDGF-hAPP model.
In contrast to the hAPP splicing observed in the PDGF-hAPP model, the hAPP splicing pattern in the cerebral cortex of mice expressing the entire hAPP gene via yeast artificial chromosomes resembled that of the endogenous hAPP gene in humans, with transgenic mice showing substantially higher ratios of APP751/APP695 mRNA levels than nontransgenic controls and low levels of hAPP770 expression(41) . This indicates that the higher levels of APP751 mRNA expression in humans compared with mice is probably related to regulatory sequences within the hAPP gene itself rather than to differences in the neuronal splicing machinery of humans versus mice.
Although the PDGF-hAPP transgene is also expressed in
peripheral organs, PDGF B chain promoter-driven gene expression within
the CNS is restricted predominantly to neurons ( (42) and data
not shown). Hence, the unusual alternative splicing of PDGF-hAPP
pre-mRNA (Fig. 6B and Table 2) suggests that the
normal neuron-specific skipping of APP exons 7 and 8 in vivo depends, at least in part, on the hAPP intronic sequences that
were modified in the construction of this fusion gene (Fig. 1C). It is conceivable that these sequences
normally participate in the regulation of splice site selection, for
example, by interacting with cell-specific RNA-binding proteins that
may block the use of certain exons(43) . Notably, studies on
the -tropomyosin pre-mRNA have demonstrated that alternative
splicing patterns can be altered not only by deletions of intron
sequences but also by clustered point mutations within introns that do
not alter the distance between 3`-splice sites and upstream
branchpoints(43) . Hence, while it is tempting to speculate
that the large deletions within hAPP introns 6 and 8 had a greater
effect than the XhoI site elimination in hAPP intron 7, it is
possible that both types of modifications contributed to the difference
in alternative splice site selection and hAPP exon usage observed
between the PDGF-hAPP and endogenous APP pre-mRNAs. Because there is
evidence suggesting that APP splicing within neurons can be regulated
by factors that influence neuronal differentiation and
activity(44, 45, 46, 47, 48, 49) ,
it should be noted that no obvious abnormalities in CNS development
have been identified in PDGF-hAPP mice and that their neurons show no
morphological evidence of dedifferentiation (data not shown). We
therefore believe that the hAPP intron modifications discussed above
provide the most plausible explanation for the hAPP770/751-predominant
splicing pattern identified in this model.
As an aside, our comparison of PDGF-hAPP and endogenous human genomic DNA (Fig. 1, C and D) revealed that the authentic hAPP intron 7 is actually shorter than indicated by a previous study (34) . As outlined under ``Results,'' the previously published map can be reconciled with our data if one assumes that two neighboring EcoRI fragments of the hAPP gene, one of which contains exon 8, are arranged in an order opposite to the one reported by Yoshikai et al.(34) .
Does a shift toward the
expression of KPI-containing hAPP play a causal role in
amyloidogenesis, and can similar alterations be identified in humans
with AD? As summarized in Table 3and Table 4, the
pertinent literature is complex. Many of the controversies in this
field probably relate to specific differences among studies with
respect to stages of AD selected, specific CNS regions examined,
integrity of RNA samples analyzed, and RNA detection methods used. When
analyzing RNA extracted from autopsy material, it is frequently
difficult to ensure that alterations in the levels of specific
transcripts reflect effects of the disease state under study and not of
the death process or postmortem alterations(50, 51) .
Our RPA analysis of relatively well-preserved RNA extracted postmortem
from the midfrontal gyrus of humans with or without AD revealed no
significant change in total hAPP mRNA levels in patients with moderate
to severe AD compared with normal controls (Fig. 3C)
and a subtle increase in the relative abundance of hAPP751 mRNA (Table 2). While this study was not designed to differentiate
whether this shift reflects an increase in neuronal versus glial hAPP751 mRNA expression, others have reported that reactive
glia in AD brains show no clear overexpression of APP
mRNAs(17, 26) . It should also be noted in this
context that our hAPP770 probe does not differentiate between
transcripts encoding APP751 or amyloid precursor-related
protein-563(52) ; the latter closely resembles APP751 in its
N-terminal portion but does not encode A as it differs
substantially from APP isoforms in its C-terminal region(52) .
Amyloid precursor-related protein-563 has been found to be increased in
AD brains by reverse transcriptase-PCR and in situ hybridization(53) .
It has been postulated that the
relative increase in KPI-containing APP isoforms in the aging human
brain may increase the risk of amyloid formation (19, 54, 55, 56, 57) and
that the rarity of amyloidosis in the aged rodent brain and the
cerebellum of higher mammals is related to the low levels of
KPI-containing APP isoforms in these tissues(20) . It has
further been speculated that an overexpression of KPI-containing APP
could disturb the balance between biosynthesis and degradation of APPs
in the brain and lead to
amyloid
formation(21, 57, 58) . The pronounced
overexpression of KPI-encoding hAPP mRNAs and development of prominent
AD-type pathology in the PDGF-hAPP model might be consistent with these
postulates. However, to determine the relative neuropathogenic
potential of individual hAPP isoforms more conclusively, transgenic
models should be compared in which the PDGF promoter is used to
overexpress selectively hAPP695, hAPP751, or hAPP770 at levels similar
to those achieved in the original PDGF-hAPP mice; the generation of
such models is underway.
In contrast to the PDGF-hAPP model, transgenic mice expressing the entire hAPP gene via yeast artificial chromosomes (41, 59, 60) have apparently so far failed to develop prominent AD-type pathology. Although the difference in hAPP splicing pattern in these distinct models could contribute to the difference in neuropathological readout (see above), the presence/absence of amyloidogenic mutations in the hAPP molecules expressed and differences in the overall levels of hAPP expression may be of equal or even greater pathogenetic importance. Notably, the overall hAPP mRNA levels in hAPP yeast artificial chromosome mice do not appear to have been significantly higher than those found in human brains(41, 59) .
In conclusion, the differences in APP isoform-specific mRNA levels identified among humans and nontransgenic and PDGF-hAPP mice suggest that the neuron-specific skipping of APP exons 7 and 8 depends, in part, on the hAPP intron sequences that were modified in the construction of the PDGF-hAPP transgene. In addition, the results of the current study indicate that the development of prominent AD-type pathology in PDGF-hAPP mice could be related to (i) the substantial level of hAPP overexpression achieved in this model, (ii) the relatively high proportion of mutated hAPPs versus wild-type mAPPs expressed in their brains, and (iii) the prominent shift toward the expression of KPI-encoding APP mRNAs. While these findings strongly support a causal role of hAPP gene products in the development of AD-type brain alterations, frontal cortex tissue from humans with AD displayed only a subtle increase in the relative abundance of hAPP751 and no significant increase in overall hAPP levels compared with normal controls. However, to condense a disease process that probably takes decades to develop in humans into a substantially shorter time period, potential etiologic factors, such as APP(derivatives), may have to be expressed at significantly higher levels than those encountered in the human disease. This postulate is supported directly by the development of AD-type pathology in the high expressor PDGF-hAPP model (1) and indirectly by the earlier development of AD-type pathology in persons with Down's syndrome (5) who show roughly 1.5-fold higher levels of cerebral hAPP mRNA expression than controls(3, 4) . In addition, evidence is accumulating that the development of AD may involve complex interactions between different pathogenetic pathways, including alterations in the function and/or expression of glia-derived factors such as apoE(61, 62, 63, 64, 65, 66, 67) , cytokines(68, 69, 70) , and extracellular matrix components(71, 72, 73) . The further in vivo dissection of the pathogenetic potential of specific hAPP isoforms, derivatives and mutations as well as of their interactions with such co-factors constitutes a promising field for future investigations.