From the Neurobiology Research Lab, Heartland
Veterans Integrated Service Network, Kansas City, Missouri 64128, the § Departments of Neurology, ¶ Pathology and
Laboratory Medicine, and ** Pharmacology, Toxicology, and Therapeutics,
the University of Kansas Medical Center,
Kansas City, Kansas 66160, and
Department of
Integrative Biology, Pharmacology, and Physiology, University of Texas,
Houston Health Science Center, Houston, Texas 77225
Received for publication, June 1, 2000, and in revised form, September 14, 2000
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ABSTRACT |
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In order to understand the mechanism for
insoluble neurotoxic protein polymerization in Alzheimer's disease
(AD) brain neurons, we examined protein and gene expression for
transglutaminase (TGase 2; tissue transglutaminase (tTG)) in
hippocampus and isocortex. We found co-localization of tTG protein and
activity with tau-positive neurofibrillary tangles, whereas mRNA
and sequence analysis indicated an absolute increase in tTG
synthesized. Although apoptosis in AD hippocampus is now an established
mode of neuronal cell death, no definite underlying mechanism(s) is
known. Since TGase-mediated protein aggregation is implicated in
polyglutamine ((CAG)n/Qn expansion) disorder apoptosis,
and expanded Qn repeats are excellent TGase substrates, a role
for TGase in AD is possible. However, despite such suggestions almost
20 years ago, the molecular mechanism remained elusive. We now present
one possible molecular mechanism for tTG-mediated, neurotoxic protein
polymerization leading to neuronal apoptosis in AD that involves not
its substrates (like Qn repeats) but rather the unique presence
of alternative transcripts of tTG mRNA. In addition to a
full-length (L) isoform in aged non-demented brains, we found a short
isoform (S) lacking a binding domain in all AD brains. Our current
results identify intron-exon "switching" between L and S isoforms,
implicating G-protein-coupled signaling pathways associated with tTG
that may help to determine the dual roles of this enzyme in neuronal life and death processes.
Transglutaminases
(TGases,1 EC 2.3.3.13) are a
gene family of transamidating enzymes that, under the influence of
calcium, catalyze protein cross-linking through acyl transfer of
specific glutamine residues to lysines. These enzymes are involved in a variety of key metabolic processes that range from blood coagulation to
cell death. Expression for the most ubiquitous intracellular member,
tissue TGase (tTG), is highly regulated. It is induced in cultured
cells by various agents including cytokines, such as interleukin-6
(IL-6) (1, 2), cyclic AMP (3-5), activation of the transcription
factor, NF In this context, the history of tTG in Alzheimer's disease (AD)
pathogenesis began almost 20 years ago with the report that brain tTG
catalyzed cross-linking of neurofilament molecules (18). A decade
later, A In the last few years, tTG was shown to be a bifunctional enzyme, a
G-protein possessing GTPase activity (26, 27) capable of binding GTP
(and ATP) (28-30), in addition to its cross-linking activity. This
also suggests dual roles in programmed cell life as well as death. GTP
binds at the COOH-terminal of the full-length protein (27-32), which
is designated G We reasoned that if injury could rapidly induce S isoforms that are
more associated with apoptotic cell death, a search for similar splice
variants in neurodegenerative diseases might be revealing. Therefore,
we examined cortical and hippocampal regions of AD patients and
non-demented matched controls for tTG, isodipeptide bonds, TGase
enzymatic activity, and gene expression for the full-length tTG gene
(TGM2) in parallel with tau in NFTs. Finally, using specific oligonucleotide primers, we determined which mRNA transcripts were
produced in AD, and we compared these with non-demented, aged brains.
Our results confirm several recent reports that TGase enzymatic
activity is increased in hippocampal regions of neuropathologically confirmed AD brains (25, 39). We further found that increased activity
correlates with increased expression of TGM2 in these same
brain regions. Finally, we report the novel finding in human tissue
that alternative mRNA processing for TGM2 occurs in AD. This alternative splicing is accompanied by protein precipitation in
the form of insoluble inclusions, in situ isodipeptide bond formation, and increased TGase activity
levels.3
Materials--
Brain samples were obtained from the Alzheimer's
Disease Center Neurospecimen Brain Bank at the University of Kansas
Medical Center. All AD brains came from patients with the clinical,
ante-mortem diagnosis of AD. The 9 AD brains used in these studies
ranged in age at death from 69 to 93 years, except for one 51-year-old patient with both trisomy 21 and AD. Numerous neuritic plaques and NFTs
were identified in modified Bielschowsky-stained sections of isocortex
as well as hippocampus. The neuritic plaques were in sufficient
quantities for the diagnosis of AD using Consortium to Establish
a Registry for Alzheimer's Disease criteria (40, 41). Controls came
from age-matched, non-demented individuals. All samples for biochemical
studies had been rapidly frozen in liquid nitrogen within 8 h of
death and preserved at
Oligonucleotide primers were synthesized at the University of Kansas
Medical Center Biotech support facility. Bicinchoninic acid kit was
purchased from Pierce. Guinea pig liver tTG,
N,N-dimethylcasein, and diaminobenzidine (DAB) chromogen
were bought from Sigma. [3H]Putrescine
(7.77·1012 dpm/mol) was obtained from New England
Nuclear. Trizol reagent was bought from Life Technologies, Inc. PCR mix
was from PerkinElmer Life Sciences; reverse transcriptase, Superscript
II, was from Life Technologies, Inc. Antibody to tTG was purchased from
the Neomarkers Division of Lab Vision Corp. (Fremont, CA). Rabbit anti-human tau polyclonal antibody was from Dako (Carpenteria, CA) as
was a labeled streptavidin-biotin kit used for visualization. Monoclonal antibody that recognizes the
N- Analysis of AD Versus Control Brain tTG
Immunocytochemistry--
Both frozen and fixed tissue reacted equally
well with tTG and isodipeptide antibodies. The results and
photomicrographs reported here are from formalin-fixed and
paraffin-embedded tissue that were deparaffinized and microwaved in
citrate buffer for antigen retrieval. For double labeling experiments,
the sections were formalin-fixed and embedded in paraffin. The primary
antibodies were applied sequentially; the first antibody was a rabbit
anti-human tau, made against four repeated sequences involved in
microtubule binding from the COOH-terminal part of the human tau
protein (43). The secondary antibody was a biotinylated anti-rabbit
IgG, and this was followed by streptavidin conjugated to alkaline
phosphatase and revealed by Fast Red as chromogen for visualization.
The tTG antibody was a mouse monoclonal antibody made against purified guinea pig liver TGase 2. A secondary biotinylated anti-mouse antibody
was followed by streptavidin labeled with horseradish peroxidase. The
DAB chromogen used for horseradish peroxidase visualization gave a
brown precipitate.
Fluorescence microscopy was performed with 7-µm
formalin-fixed, paraffin-embedded sections. Mouse monoclonal anti-tTG
was used at 1:100 dilution and visualized with fluorescein
isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary
antibody. Controls included preimmune serum in place of the primary
antibody. The mouse monoclonal antibody to isodipeptide bonds was used
at a 1:100 dilution and was visualized with goat anti-mouse secondary antibody conjugated to CY3 (42, 44).
Fluorescence was captured on a Nikon Eclipse TE-300 fluorescence
microscope linked to a Bio-Rad MicroRadiance Plus confocal system
utilizing HyQ filters for direct observation on the microscope and 543 and 488 nm laser lines on the confocal system for CY3 and FITC,
respectively. In some experiments, stained sections were also viewed on
a Leitz Orthoplan microscope under epifluorescence using Ploem filters.
Double-stained sections were photographed on a Nikon UFX automatic
camera and digitized, and images were collected and analyzed on a
Macintosh G3 computer using Adobe Photoshop 4.0.
TGase Enzymatic Activity Assay--
Brain tissue samples
weighing 35-55 mg were homogenized in 10 ml/g of 10 mM
Tris-HCl buffer, pH 7.4, containing 250 mM sucrose, 1 mM EDTA, 4 mM dithiothreitol, 125 mM potassium thiocyanate, 1% Lubrol-PX, with a Polytron
(Brinkman, Westbury, NY) at 4 °C 3 times at 5-s bursts. Protein
concentrations were determined with a micro-bicinchoninic acid assay
(45). TGase activity was determined by a modified assay (46, 47) in
which brain protein extracts (50-µl aliquots) were added to 0.7 ml of
50 mM Tris-HCl, pH 7.4, containing 1.3 mM
CaCl2, 10 mM dithiothreitol, 5 mg/ml N,N-dimethylcasein, 0.4 mM
[3H]putrescine and incubated for 150 min at 37 °C,
after confirmation of linearity over the period. Gunea pig liver tTG
was used for the standard curve. The incorporated putrescine was
measured by precipitation with 20% trichloroacetic acid, resuspension
in NaOH, and scintillation counting of triplicate samples in a Packard 2200CA Tri-Carb Liquid Scintillation Analyzer. Negative controls included reactions without substrate, and blanks were without enzyme source.
mRNA Analyses--
RNA was extracted (48) with denaturing
phenol (Trizol reagent). Tissue was powdered under liquid
N2 and homogenized on ice with a Polytron. Nucleoprotein
complexes were dissociated at room temperature for 5 min and then 0.2×
volume of chloroform was added, and the solution was first agitated for
15 s and then incubated at room temperature for 3 min. The aqueous
phase, obtained after centrifugation at 15,000 × g for
15 min at 4 °C, was precipitated with 2-propanol, washed with 75%
ethanol, and resuspended in H2O. The RNA concentration was
determined by absorbance at 260 and 280 nm and electrophoresis in
formaldehyde-agarose gels.
RNA samples were first reverse-transcribed to produce cDNA with the
downstream primer (5'-TGGTAGATGAAGCCCTGTTG). These were performed in 20-µl reactions with 1 µg of total brain RNA, 1 mM dNTPs, 1× PCR mix, 10 pmol of primer, and 200 units of
reverse transcriptase (Superscript II). Primers were annealed at room temperature for 10 min followed by cDNA synthesis at 42 °C for 30 min, then 5 min denaturation at 95 °C, and then quick cooling. The cDNA was amplified in PCR brought to 100 µl containing a
total of 50 pmol of each of the upstream (5'-GCTGCTCCTGGAGAGGTGTG) and downstream primers and 200 µM dNTPs. The reactions were
first denatured at 98 °C for 10 min, cooled to 90 °C for addition
of 2.5 units of Taq polymerase (Amplitaq, PerkinElmer Life
Sciences or Biolase, Bioline), and followed by 30-35 cycles of
95 °C for 1 min, 55 °C for 1 min, and 72 °C for 45 s.
Products were visualized under UV light after agarose gel
electrophoresis and ethidium bromide staining, and band intensities
were measured by direct CCD camera capture and analysis with NIH Image
(49).
Real Time Quantitative RT-PCR (qRT-PCR)--
To quantify tTG
(TGase 2) mRNA levels, we turned to a method previously exploited
to quantify message in liver (50). We measured the displacement of
fluorescently labeled and quenching dye-labeled internal
oligonucleotides by the PCR amplification and a real time system (ABI
Prism 7700, Applied Biosystems, Foster City, CA) used according to
manufacturer's recommendations.
tTG mRNA Sequences--
Amplifications with primers spanning
the entire coding region as well as primers directed toward the
COOH-terminal region were employed to analyze for alternative tTG
transcripts in AD versus non-demented control brain
mRNA. The primers utilized in these experiments included the
upstream primer (5'-GGAGCCAGTTATCAACAG) and the downstream
primer (5'-GGGTTATAAATGGAGCAG). The RT-PCR amplifications were
performed as described above.
Amplimer Sequencing--
To identify unambiguously the potential
alternative tTG transcripts, we isolated RT-PCR products, removed
primers with two ethanol precipitations, and sequenced these on an ABI
fluorescent automated sequencer, Prism model 377. Nested primers were
included for the sequencing reactions, or RT-PCR products were
subcloned into pCRII utilizing the TOPO T/A cloning kit (Invitrogen,
Valencia, CA) according to the manufacturer's directions, and plasmids
were purified with a spun column miniprep procedure (Qiagen, Carlsbad, CA). Data were examined with Applied Biosystems Editview software and
the University of Wisconsin Genetics Computing Group Software (51).
tTG Immunolocalization in Control and AD Brain--
To establish
the distribution of tTG in our AD patient samples (ranging in age from
65 to 81, n = 5), brain sections were characterized
using tTG, and tau immunohistochemistry sections examined included the
hippocampus at the level of the lateral geniculate nucleus, cingulate
gyrus, superior temporal lobe, mid-frontal gyrus, and striate cortex.
All of the AD patients showed severe NFT formation in all sections
examined except the occipital cortex consistent with Braak and Braak
stage 5 out of 6 (52). Using tau immunohistochemistry, NFTs were not
seen in any location in the control brains. Frequent neuritic plaques
were also present in all sections of cortex examined, and these were in
sufficient quantities to confirm the clinical impression of AD using
Consortium to Establish a Registry for Alzheimer's Disease criteria.
With tTG immunohistochemistry, staining of blood vessels was present in
all brains; of interest, vascular staining was less prominent in AD
brains. In stark contrast, neuronal staining was only seen in the AD
brains. The most obvious tTG-positive staining was present in the
hippocampus and adjacent regions (subiculum, transentorhinal, and
entorhinal cortex). In addition, with tTG antibody many pyramidal
neurons showed fine granular staining, whereas a minority showed
additional weak diffuse staining. Isocortical neuronal tTG staining was
also present in each AD brain examined, and again, this was most
obvious in the large pyramidal neurons. As in hippocampus, isocortical
immunostaining was occasionally granular but more often weak and
diffuse. Although one control brain showed weak tTG staining in the
lateral geniculate nucleus, no definite hippocampal or isocortical tTG
immunostaining was present.
Fig. 1 shows that hippocampal brain
regions of AD patients contained significant numbers of diffuse and
neuritic plaques, numerous intraneuronal NFTs (Fig. 1, C and
D, small arrows), significant cell loss, and gliosis.
Antibody to tTG, visualized by the brown DAB stain, was detected in
both hippocampal regions (Fig. 1, C and D,
arrowheads) as well as in the cortex (Fig. 1, A
and B, arrowheads). Differences in tTG
immuoreactivity between AD and control brains were detected, since both
endothelial cells (Fig. 1A, linear small arrows) and neurons
stained in AD, whereas staining was exclusively in endothelial cells in
control brains (not shown).
tTG Is Associated with Cross-links in Tau-positive Brain
Regions--
The next important question concerned whether tTG was
functioning as a cross-linking agent at these brain locations. As
evidence that the localized tTG in these AD neurons was active in
situ to cross-link one or more neuronal proteins, novel
immunohistochemical evidence of isodipeptide bond formation was
detected in regions showing increased immunoreactive tTG (Fig.
2A). As found with tTG, in
some neurons isodipeptide immunoreactivity was in the form of diffuse
neuronal staining (Fig. 2A, arrowhead), but in other hippocampal neurons granulovacuolar material was stained (Fig.
2A, arrows). Significantly, in no non-demented brain could significant anti-isodipeptide bond staining be found (Fig.
2B).
Fig. 2 shows neurons with in situ isodipeptide bonds in the
AD-affected brain regions, which were also positive for tau (Fig. 1).
As shown in the figure, double labeling enabled the determination that
isodipeptide bonds were co-localized with intraneuronal inclusions (NFTs). To confirm this further, we examined fluorescently labeled samples with laser scanning confocal microscopy. Through fluorescent staining techniques, we were also able to confirm co-localization (Fig.
3) of isodipeptide bonds (Fig.
3D) with tTG antigen (Fig. 3C), and these
occurred along with intraneuronal inclusions (NFTs, Fig. 3,
A and B, arrows) in these tau-positive
regions.
Increased tTG Cross-linking Activity in AD Brains--
In support
of these immunocytochemical results, we found that tTG cross-linking
enzymatic activity was increased in brain hippocampal regions of AD
patients (53, 54) (Fig. 4), confirming a
previous report (25). Enzymatic activity was increased 1.5-8 fold in
these AD brains compared with non-demented controls (Fig. 4), with a
statistically significant p value (<0.04). Of interest, relative to unaffected AD brain regions, AD brain hippocampus and
cortex had elevated tTG activity by a factor of 3.5 ± 1.2.
tTG Gene Expression in AD Brains--
It still remained to be
shown to what extent alterations in tTG expression would account for
the elevated cross-linking activity. Activity measurements cannot
distinguish among the genes now known to transcribe proteins with TGase
activity (55). This is particularly relevant given the recent report of
TGase 1 and 3, in addition to tTG, in human brain (39). Consequently,
we employed specific primers to examine message corresponding to TGase
C/TGase 2 (intracellular tTG), encoded by the gene TGM2
(56), and confirmed the amplified products by DNA sequence analysis.
Brain cortical RNA extracts were examined by RT-PCR analysis (Fig.
5), and appropriately sized products were
obtained corresponding to tTG. Although intended to characterize the
presence or absence of the TGM2 message, the reactions were
conducted to subplateau levels by testing amplifications with different
parameters including cycle numbers. In these semi-quantitative experiments, we found that elevated mRNA levels from this gene (Fig. 5) followed the activity levels we had seen (Fig. 4). The highest
mRNA values, normalized to the product seen in control brains
(100%), were 5-fold higher. Overall, these RNA levels were elevated
3.9-fold with a p value of 0.03.
Real Time Quantitative RT-PCR (qRT-PCR)--
With the
electrophoretic analyses of mRNA levels indicating significant
increases in the AD brains, we then determined the quantitative changes
in tTG with a precise system monitoring tTG sequence present at every
cycle. With the ABI Taqman system, we used two human tTG RT-PCR primers
with an internal human tTG primer conjugated to a fluorescent probe.
Included in the reaction was a quenching molecule such that extension
of template would include nucleolytic separation of the fluorescent
primer releasing it and resulting in a fluorescent signal proportional
to the amount of template present at each cycle of amplification. In
this manner, tTG mRNA levels were determined for RNA extracts from
the frontal cortex for three age-matched controls and four AD brains.
The relative tTG mRNA per total RNA present was normalized to the non-demented aged control samples giving them a value of 1.0 ± 0.16 (S.D.), and the tTG mRNA levels were elevated 4.1 ± 1.31-fold (p = 0.011, Student unpaired t
test) in this specific assay (Fig. 6).
Alternatively Spliced tTG Variants in Human Brain Tissue--
With
this increased expression of TGM2 in AD compared with
non-demented control brains, we next asked whether alternative transcripts such as those seen in cytokine-treated rat brain astrocytes (33) and injured spinal cords2 were also present in AD
brain tissue. In order to detect alternate forms specifically affecting
COOH-terminal regulatory regions of tTG, we designed a specific primer
set spanning the 3' end of the human coding region, based on published
rat sequences (33).2 With these human-specific primers, we
analyzed the products derived from cortical samples of non-demented and
AD brains. In this manner, we could only detect long form in
non-demented brain RNA. In contrast, in AD brain samples we were able
to detect both long (L) and short (S) isoforms of tTG (Fig.
7), with tTG-specific primers spanning the COOH-terminal region of human tTG. This intriguing result suggested
that like traumatic central nervous system injury,2
neurodegeneration induced tTG alternative transcription.
Sequencing of Apparent Alternatively Spliced AD tTG--
To
identify unambiguously these potential alternatively spliced tTG
variants, we isolated amplimers from the RT-PCRs, grew them in
Escherichia coli, isolated plasmids, and sequenced these DNAs to determine the structure of the divergent COOH-terminal region.
The sequences in the divergent regions are shown in Fig. 8. The widespread full-length tTG long
form (GenBankTM accession number M55153) is present
both in aged, non-demented cortex and in the AD cortex. In contrast, in
AD cortex, but not in the aged, non-demented brain, we found evidence
for variant tTG messages (Fig. 7). This human AD short tTG mRNA
results from alternative splicing at the same position (Fig. 8),
involving a substitution immediately after amino acid residue 538, previously observed in human erythroleukemia (HEL) cells (57). In one
case (Fig. 8, italics) a slight variation in the splicing
event resulted in a different coding frame which also terminated within
60 bases of the junction.
We have demonstrated that tTG protein and activity accumulate with
tau-containing NFTs in affected regions in AD brain. The evidence
indicates that tTG is up-regulated and subsequently participates in
neuronal apoptosis in AD not through its action on substrates (like
Qn repeats), but rather through the unique presence of
alternative transcripts of tTG mRNA. Analysis of RNAs from affected
AD tissues demonstrates that elevated levels, including intron/exon
switching that produce a shortened S isoform of tTG, are commonly
found. In contrast to AD brain, S isoform message for tTG is not found
in aged, non-demented human brain to any extent. The lack of a GTP
binding domain on the S isoform implicates G-protein-coupled signaling
pathways associated with tTG that may help to determine the dual roles
of this enzyme in neuronal life and death processes.
Previous studies have established that tTG is capable of cross-linking
neuronal proteins such as NFs (18), A Our novel results with isodipeptide bonds in the human brain build on
earlier reports of the presence of isodipeptide bonds in
situ in cultured neurons (59), as well as in mutant rodent central
nervous system (60). Relevant also to our current AD results, a recent
report showed that patients with progressive supranuclear palsy also
express tau proteins in brain with significant tTG-catalyzed
isodipeptide bonds (61). Furthermore, insoluble tau found in the AD
brain also harbor these isodipeptide bonds, and human tau can be
cross-linked by tTG in vitro (62, 63). The detection of
these Thus, TGase 2/tTG gene expression is clearly increased in brain regions
affected by the brunt of the disease process in AD. In support of this
finding, a recent report indicated differential expression of TGases
1-3, with increased expression for both TGase 1 and 2 (tTG) in AD
brain (39).
What might be the basis for this up-regulation? Although experiments to
address this in AD are in progress, it is known that retinoic acid
up-regulates tTG, acting through one of several retinoic acid receptors
and response elements (3, 11, 12, 65, 66). Other transcription factor
recognition sites are present in the promoter regions such as AP-1 and
SP-1 (67). In development, Tgm2 expression appears to be
essential for "appropriate" apoptosis (68-72). Since apoptotic
neuronal loss has been documented in AD and related disorders (34-37),
"inappropriate" AD-associated apoptotic neuronal death may be due,
at least in part, to up-regulation and increased activity of tTG (73).
It might also relate to changes in the dual function of the
tTG/G The dual enzymatic functions of tTG may be essential to its divalent
action in cellular life as well as death (26, 28-30, 44, 74, 75).
GTP-independent tTG may be more involved with apoptotic neuronal death
as evidenced from studies of trophic deprivation in primary rat
forebrain cells in culture (44). When ligand is provided, coupling of
the Such evidence for alternative tTG transcripts as we present here has
not previously been detected in human tissue. In fact, our report of S
form in injured spinal cord is the first example in
vivo.2 However, a TGase homologue was earlier
described in HEL cells (79), and then a third was reported (57), which
also was found to hydrolyze GTP. The latter finding suggests that this
form is more homologous to L than to S isoforms; although when
expressed in E. coli, this homologue had a
Km value several orders of magnitude less than the L
form for binding GTP (26). More importantly, the human AD tTG short
form we have identified results from alternative splicing at precisely
the same intron/exon boundaries first seen in these HEL cells (79).
This alternative message results from a substitution of intron X for
exon 11 (80), a differential processing analogous to the alternate
splicing seen in cultured rat astrocytes treated with either IL-1 Strong evidence implicates intraneuronal protein aggregation in
the pathogenesis of the CAG (polyglutamine tract) repeat diseases, which like AD also exhibit late onset neuronal death (82, 83). These
CAG diseases all involve different genes whose specific gene products
exhibit long polyglutamine repeats that are excellent substrates for
tTG cross-linking (83-86). In Huntington's disease, for example,
neuronal nuclear inclusions are the result of elevated tTG and
tTG-catalyzed aggregations as evidenced by tTG cross-links (87).
Protein aggregation in neurodegeneration has recently been extended to
include AD and related disorders in which long polyglutamine repeats
are not present and in which cross-linking may be due to some other
feature of the protein, such as point mutation, oxidative injury, or
misfolding and altered chaperone function (88-91) and/or, as we
suggest, alternative tTG RNA processing.
The definitive test for the role of G The elevated TGM2 mRNA levels in AD brain indicate that
enhanced TGase cross-linking activity is most likely due to increased synthesis of the enzyme rather than post-translational processing. The
increase, and/or altered transcription, in mRNA levels suggests that tTG elevation may be a good predictor, a surrogate marker, for the
pathophysiology underlying the diagnosis of AD. In this regard,
identification of the critical protein substrates cross-linked by tTG
in the brain may also help to uncover central targets for therapeutic
intervention early in the course of AD (82, 96). Further understanding
of the regulation of TGM2 transcription (11, 12, 38, 66) may
provide additional opportunities to control tTG expression as a
potential therapy against inappropriate apoptosis in AD and related
neurodegenerative disorders.
Conclusions--
By analysis of protein immunoreactivity,
enzymatic activity, and mRNA levels, we have shown that tTG is
elevated in AD brains and co-localizes with tau in NFTs, prominent
inclusions that are one of the hallmarks of neuronal degeneration. This
elevation approaches 5-fold compared with non-demented aged brain, is
most dramatic in association with NFTs stained with tau in AD brains, and not at all in control brains. We have demonstrated for the first
time that these tau-containing NFTs are the site of
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ABSTRACT
INTRODUCTION
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DISCUSSION
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B (6-8), and DNA methylation (9). The most potent
inducers of tTG gene expression are retinoids (10-12), which also
promote apoptosis in various cells (13, 14), including neurons
(15-17).
peptide (19, 20) as well as the
-amyloid precursor
protein (21) were shown to be cross-linked by tTG. Subsequently, tTG
protein was demonstrated within amyloid plaques in AD brains (22). By
using immunohistochemistry and an antibody to coagulation factor XIII,
an extracellular TGase that cross-reacts with tTG, co-localization with
paired helical filaments, the major components of neurofibrillary
tangles (NFTs) in AD neurons were reported further suggesting a role
for tTG in AD pathogenesis (23). Consistent with this notion, the
phosphorylated microtubule protein, tau, a molecular component of NFTs
and paired helical filaments, associates with tTG to form insoluble
filaments (24). In recent studies, tTG activity was found to be
increased in the most severely affected brain region in AD, the
hippocampus (25).
h/tTG. An important discovery was the
appearance of an alternatively spliced short (S) form lacking the
GTP-binding site when rat brain astrocytes in culture were treated with
the cytokines interleukin 1
(IL-1
) or tumor necrosis factor-
.
With translation of this S form, cross-linking activity would no longer
be negatively regulated by GTP (33). This loss-of-function might impact
on AD and other neurodegenerative diseases, where apoptosis is
prominent (34-37). This finding led us to consider first whether
alternatively spliced variants were produced in response to injury. We
have found evidence of such alternative transcripts in rat spinal cord
within the first 8 h after controlled contusion
injury.2
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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70 °C. Sections for immunohistochemistry
were processed routinely with formalin fixation and paraffin embedding.
(
-glutamyl)lysine isodipeptide bonds produced
by the action of TGase activity (42) was 811-MAG purchased from Covalab
(Lyon, France).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Co-localization of tau and tTG in
neurofibrillary tangles. Frontal cortex (A and
B) and hippocampus (C and D) tissue
samples from a severely involved case of Alzheimer's disease were
sectioned and incubated with anti-tau visualized with Fast Red (red
chromagen) and anti-tTG visualized with DAB (brown
chromagen). In the cortex (A), tau immunostaining
(arrows) is evident primarily within pyramidal neurons in
layers 3 and 5, and a higher magnification image is also shown
(B). Most tau-stained neurons also stain positively with
anti-tTG. Some tTG staining was also visible in capillaries and in
granular/granulovacuolar neurons. In the hippocampus (C),
anti-tau stained residual pyramidal cells, and double immunostaining
with tTG showed that many tangle (NFT)-containing cells (example
magnified in D) also labeled with tTG
(arrowheads). In addition, tTG staining was also found in
capillary endothelial cells (linear small arrows),
cross-reacting with factor XIII, but was much reduced in glial
cells.
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Fig. 2.
Isodipeptide immunolocalization in human
brain samples. By using an antibody to anti-isodipeptide bonds,
pyramidal neurons in the CA1 of AD brain hippocampus sections
immunostained in a focal pattern that resembled granulovacuolar
degeneration (GVD; B, small arrows). Some neurons
were also diffusely stained (arrowhead). This positive
isodipeptide staining did not appear in age-matched normal brain
sections (A).
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Fig. 3.
Anti-tTG and anti-isodipeptide bond
immunostained AD tissue from the cortex area. By using modulation
contrast (Hoffman) microscopy (A and B), visible
neurons containing NFTs (arrows) stained positively with
both anti-tTG (visualized with FITC, C) and
anti-isodipeptide (visualized with Texas Red, D)-conjugated
secondary antibody under confocal microscopy.
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Fig. 4.
tTG activity measurements in human brain
samples. AD and age-matched control brain samples
(A-D) were obtained and assayed for tTG activity as
described under "Experimental Procedures."
View larger version (55K):
[in a new window]
Fig. 5.
RT-PCR analysis of tTG mRNA in human
cortex. Total RNA, from normal and AD samples in Fig. 2,
was reverse-transcribed and amplified by PCR to subplateau levels with
tTG-specific primers and separated by agarose gel electrophoresis
(B) as under "Experimental Procedures." Ratios between
the products obtained produced relative tTG mRNA levels summarized
in A.
View larger version (16K):
[in a new window]
Fig. 6.
Quantitative RT-PCR for cortical tTG mRNA
levels in AD brains. We quantitatively determined the increase in
tTG message seen in AD relative to aged-matched controls as described
under "Experimental Procedures."
View larger version (17K):
[in a new window]
Fig. 7.
Variant tTG messages present in AD
brains. By using human-specific primers spanning the GTP binding
region, in TGM2 and designed to identify L and S isoforms,
we examined tTG messages present in normal and AD-affected brain
samples by RT-PCR (RT), as described under "Experimental
Procedures." RNAs from AD brains contained greater amounts of tTG S
form relative to L form when compared with non-demented aged control
(Normal) brains.
View larger version (25K):
[in a new window]
Fig. 8.
Alternate splicing in AD brains. The
protein (A) and DNA (B) sequences for the tTG
short forms found in AD patient brains are compared with the
age-matched control. The domains surrounding the sequence divergence in
the COOH-terminal regions are shown. Coordinates are amino acid
positions and base positions corresponding to the long form sequence.
Translated DNA sequences are underscored, and termination
codons are boxed in the cDNA sequence and denoted by *
in the amino acid sequence. Sequence differences to the wild-type
isoform (56) are depicted in lowercase, and differences
among the short forms observed in AD are shown in italics.
The active site at position 277 and the calcium binding domain are
unaffected by these alternative messages. However, the GTP binding
domain in the COOH terminus (arrows) is absent in all of
these AD short form coding sequences.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide (19),
-amyloid
precursor protein (21), the non-amyloid component,
-synuclein (58),
and tau (24), all implicated in the pathogenesis of AD and/or other
neurodegenerative diseases. Furthermore, using an antibody to factor
XIII, an extracellular TGase that antigenically cross-reacts with TGase
2 (tTG), tTG was detected in hippocampal neurons in AD but not control
brains (23), and TGase enzymatic activity was reported elevated in AD
brain (25). At what level transcriptional or post-transcriptional
regulation of increased tTG activity occurred in AD pathogenesis was
not addressed in those studies.
-glutamyl-
-lysine cross-links represents unambiguous
evidence for the in situ activity of one or more TGases in
the tissue where they are found (42, 64). Our present results also
confirm that tTG-specific (using an anti-TGase 2 antibody, distinct
from factor XIII) immunoreactivity is localized to neurons in
hippocampus and cortex (23). We also corroborate that TGase cross-linking activity is increased, from 2- to 8-fold, in extracts of
affected brain regions from AD patients but not in non-demented brains
(Fig. 4). In this context, we found that tTG was one of the TGases
increased since we found a semi-quantitative increase in specific TGase
2 (tTG) mRNA (Fig. 5) using specially designed primers. These
results suggest that regulation of tTG in AD brain was
pretranslational. Real-time qRT-PCR confirmed this result, as we
identified a 4-fold increase (Fig. 6) in tTG copy number per µg of RNA.
h. In this regard, the "switch" from GTPase to
unregulated cross-linking activity (29) may be critical in nervous
system degeneration (33).
1-adrenergic receptor to phospholipase C
1 (29,
76, 77) via G
h (78) promotes programmed cell life. In
contrast, when trophic ligand is deprived, a loss of peripheral
distribution of tTG protein occurs with an increase in isodipeptide
bonds, the in situ product of the TGase reaction in the
cytoplasm of cells undergoing apoptosis (44). Suggesting that
deprivation coincided with the onset of GTP-independent tTG activity,
photoaffinity labeling revealed reduced binding to
G
h/tTG in deprived cultures, whereas formation of
inositol triphosphate and mobilization of [Ca2+]i
were reduced. Thus, loss of G
h/tTG signal transduction during a period when cell survival is reduced following withdrawal of
1-adrenergic agonist supports the hypothesis that
G
h/tTG might represent a switch operating with
either programmed cell life or death and that this might result from
altered synthesis of mRNA transcripts. The switch to S isoform
might reflect a reduction in regulation that takes place at the level
of transcription.
or
tumor necrosis factor-
(33) and similarly results in the loss of a
GTP-binding domain (81). Thus, increased GTP-independent tTG might be
involved with concomitant neuronal death via an apoptotic pathway in AD brains as it is after catecholamine deprivation in primary rat forebrain cells in culture (44).
h/tTG in
neurodegeneration will likely come from total or tissue-specific
conditional mutant (knockout) studies in mice. Cloning of the mouse
G
h/tTG gene (Tgm2), homologous to
TGM2 in humans, and coding for the dual-function protein
that binds and catalyzes transamidation of glutamine residues have been
accomplished (92). However, publication of G
h/tTG
knockout mice has not yet appeared. On the other hand, mice null for
the keratinocyte Tgase (TGase 1), also recently found increased in AD
brain (39), do not exhibit neurological abnormalities but do present
severe epidermal deficiencies (93). In humans, celiac disease is an
autoimmune disorder involving/tTG and a high glutamine content protein
(gliadin) and is also associated with brain abnormalities (94, 95).
-glutamyl-
-lysine cross-links in these same locations in brains
from AD patients. This may be common to another rare neurodegenerative
disorder, progressive supranuclear palsy, where a recent report shows
these cross-links in NFT tau (61). A first clue to the marked increase in tTG activity is the unique identification in demented human brain of
tTG messages with a swapped intron-exon. Since our sequence analysis
confirms that the known inhibitory GTP binding domain in this protein
is absent in this AD alternatively spliced form, this should serve as a
potential molecular mechanism for production of neurotoxic aggregates
and neuronal cell death characteristic of AD.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Charles DeCarli and the University of Kansas Medical Center Alzheimer's Disease Center for encouragement and providing AD and control samples; Dr. Kenneth Peterson and Zhiming Suo for helpful discussion; and Sheila Price, Mary Landis, and Fan Qin for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Department of Veterans Affairs Medical Research Service (to B. W. F.), Missouri Alzheimer's and Related Disorders Fund (to B. W. F.), the University of Kansas Medical Center Research Institute, Inc. (to B. W. F. and K. S. C.), and the Midwest Biomedical Research Foundation (to B. A. C.).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: Neurobiology
Research Lab (151), Heartland Veterans Health Service Network, Veterans Affairs Medical Center, 4801 Linwood Blvd., Kansas City, MO 64128. Tel.: 816-861-4700 (ext. 7079); Fax: 816-922-3375; E-mail:
serpin@eagle.cc.ukans.edu.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M004776200
2 B. W. Festoff, C. Yong, K. SantaCruz, P. M. Arnold, C. T. Sebastian, P. J. A. Davies, and B. A. Citron, submitted for publication.
3 Citron, B. A., SantaCruz, K., Davies, P. J. A., Peng, J.-H., and Festoff, B. W., presented in abstract form at the 27th Annual Meeting of the Society for Neurochemistry, Chicago, IL, March 26-29, 2000.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TGase, transglutaminase; tTG, tissue transglutaminase; NFT, neurofibrillary tangle; IL, interleukin; AD, Alzheimer's disease; NFTs, neurofibrillary tangles; DAB, diaminobenzidine; FITC, fluorescein isothiocyanate; qRT-PCR, quantitative reverse transcription coupled to polymerase chain reaction; HEL, human erythroleukemia; RT-PCR, reverse transcriptase-polymerase chain reaction.
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