Download Transglutaminase Is Linked to Neurodegenerative Diseases

J Neuropathol Exp Neurol
Copyright Ó 2007 by the American Association of Neuropathologists, Inc.
Vol. 66, No. 4
April 2007
pp. 258Y263
REVIEW ARTICLE
Transglutaminase Is Linked to Neurodegenerative Diseases
Nancy A. Muma, PhD
Abstract
Transglutaminase catalyzes a covalent bond between peptidebound glutamine residues and either lysine-bound peptide residues
or mono- or polyamines. Multiple lines of evidence suggest that
transglutaminase is involved in neurodegenerative diseases including Alzheimer disease, progressive supranuclear palsy, Huntington
disease (HD), and Parkinson disease. In all of the neurodegenerative diseases examined to date, transglutaminase enzyme activity
is upregulated in selectively vulnerable brain regions, transglutaminase proteins are associated with inclusion bodies characteristic
of the diseases, and prominent proteins in the inclusion bodies are
modified by transglutaminase enzymes. These prominent proteins
in the inclusion bodies, including tau, >-synuclein, and huntingtin
protein, are modified by transglutaminase in vitro and >-synuclein
and huntingtin protein are modified in cells in culture. Similar
changes in transglutaminase and transglutaminase-modified proteins are replicated in transgenic mouse models of the neurodegenerative diseases, including Huntington disease and progressive
supranuclear palsy. Lastly, inhibition of transglutaminase either via
drug treatments or molecular approaches is beneficial for the
treatment of HD transgenic mice but has yet to be explored for the
other neurodegenerative diseases. Further research is needed to
determine the specific role(s) that transglutaminase plays in the
pathophysiology of neurodegenerative diseases with possible implications for transglutaminase as a therapeutic target.
Key Words: Alzheimer disease, Huntington disease, Progressive
supranuclear palsy, Parkinson disease, Transglutaminase.
INTRODUCTION
The involvement of transglutaminases in the pathophysiology of a neurodegenerative disease, namely Alzheimer disease (AD), was first suggested more than two
decades ago by Dennis Selkoe, Carmela Abraham, and
colleagues in their investigations on neurofibrillary tangles
(1, 2). Their studies demonstrated the presence of transglutaminases in postmortem human brain of normal individuals and in those with AD. Transglutaminases are inducible
From the Department of Pharmacology, Loyola University Medical Center,
Maywood, Illinois.
Send correspondence and reprint requests to: Nancy A. Muma, PhD,
Department of Pharmacology and Toxicology, University of Kansas
School of Pharmacy, 5064 Malott Hall, Lawrence, KS 66045-2505;
E-mail: [email protected]
This work was supported by Grant No. 446 from the Society for Progressive
Supranuclear Palsy.
258
enzymes, with increased activity during development,
terminal differentiation, and apoptosis (3). Increases in
transglutaminase-catalyzed ?-(F-glutamyl) lysine bonds are
found in various pathologic tissues such as atherosclerotic
plaques and tumors and in lens tissue in cataract formation
(4, 5) as well as in several neurodegenerative diseases (6Y8).
Transglutaminases (EC 2.3.2.13) are a family of
calcium-activated enzymes that catalyze the transamidation
of glutamine-containing peptides and proteins by primary
amine nucleophiles such as polyamines or small amines such
as serotonin, spermine, or spermidine (9). Transamidation
increases the stability of the protein substrates (10). Transglutaminase also catalyzes covalent ?-(F-glutamyl) lysine
cross-links between peptide-bound lysine and peptide-bound
glutamine residues of substrate proteins (for reviews, see
References 11, 12). This type of covalent bond can crosslink proteins into stable, rigid, insoluble complexes (13),
reminiscent of inclusion bodies seen in neurodegenerative
diseases. Transglutaminases can act in one of two ways to
form stabile polymers (14). Transglutaminase can direct the
de novo formation of small oligomers and polymers by
cross-linking substrate proteins such as in the formation of
copulatory plugs. Alternatively, transglutaminase can Bspotweld[ preformed oligomers and polymers reversibly
assembled via noncovalent bonds, resulting in the stabilization of the polymer. The transglutaminase, factor XIIIa,
functions in this way to stabilize the formation of blood
clots. Based on data demonstrating the assembly of dynamic
tau filaments in vitro, transglutaminase-catalyzed cross-links
may stabilize assembled tau oligomers in AD.
TRANSGLUTAMINASE-CATALYZED BONDS IN
INCLUSION BODIES
The transglutaminase-catalyzed bond has been shown
to be associated with intracellular inclusions and the proteins
contained within those inclusions in several neurodegenerative diseases, suggesting that it is a general mechanism or
common pathway involved in the pathophysiology of these
diseases. In AD and progressive supranuclear palsy (PSP),
one of the major inclusion bodies is neurofibrillary tangles,
which label with the transglutaminase-catalyzed cross-link
directed antibodies (15Y17). Purified straight and paired
helical filaments from AD and PSP tissue contain transglutaminase cross-linked tau protein as demonstrated by
immunoprecipitation and Western blots (16, 18). Furthermore, the levels of the transglutaminase-catalyzed bonds
were found to be increased with age and more than
threefold higher in AD compared with age-matched controls
in selectively vulnerable brain regions, including the
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J Neuropathol Exp Neurol Volume 66, Number 4, April 2007
hippocampus and frontal cortex, as measured by an ELISA
method (19). Isolation of ubiquitin-positive particles from
the insoluble fraction of AD brain contained tau proteins and
were found by high pressure liquid chromatography analysis
to contain a number of peptide sequences that were crosslinked by transglutaminase (19). Importantly, in AD, crosslinking of tau filaments occurs before the presence of
microscopically detectable neurofibrillary tangles, suggesting that transglutaminase plays a role in the formation or
stabilization of small tau oligomers or monomers (18, 20).
Similar evidence suggests that transglutaminasecatalyzed bonds are associated with Lewy bodies, the
major inclusion body in Parkinson disease (PD). By use of
immunohistochemistry, >-synuclein-labeled Lewy bodies
colabel with antibodies directed against the transglutaminase-catalyzed bond in PD cases and cases of dementia
with Lewy bodies (17, 21, 22). The transglutaminasecatalyzed cross-link bond comigrates on Western blots
with >-synuclein from PD substantia nigra (17, 22).
In Huntington disease (HD), perinuclear cytoplasmic
inclusions and intranuclear inclusions of mutant huntingtin
are prominent neuropathologic hallmarks (23Y27). Transglutaminase-catalyzed cross-links are present in neuronal
nuclei in selectively vulnerable brain regions in HD and the
cross-links colocalize with the huntingtin protein in
the inclusions (28, 29). HD and control brain tissue from
the superior frontal gyrus were analyzed at the confocal level
with double label immunofluorescence using an antibody
specific for the ?-(F-glutamyl) lysine cross-link catalyzed by
transglutaminase and an antibody specific for the amino
terminus of the huntingtin protein in conjunction with
4¶,6-diamidino-2-phenylindole, a dye that specifically labels
nuclei (29). In the HD cases, 4¶,6-diamidino-2-phenylindolelabeled nuclei contain aggregates of huntingtin that colocalized with ?-(F-glutamyl) lysine cross-links.
IN VITRO CROSS-LINKING AND
CROSS-LINKING IN CELL CULTURE
Several major proteins that comprise the inclusion
bodies can be cross-linked by transglutaminase in vitro. Tau
is an excellent substrate for ?-(F-glutamyl) lysine crosslinking by transglutaminase 2 in vitro (18, 30). Amine donor
and acceptor sites on human tau 23 and 40 (using the
Goedert numbering system) were identified in an in vitro
study (31). Not all of the glutamine and lysine residues in
tau could act as acceptor or donor sites. However, many of
the amine acceptor and donor sites on human tau are located
in and adjacent to the microtubule binding repeat region, a
region of the tau molecule implicated in the etiology of
tauopathies based on the abundance of disease-associated
mutations in the region. Tau protein is highly phosphorylated in AD, PSP, and other tauopathies, especially tau in the
paired helical filaments and neurofibrillary tangles. Transglutaminase can cross-link phosphorylated and nonphosphorylated tau protein (32). Furthermore, transglutaminase
can cross-link tau into straight and paired helical filaments
(33), bundles of 10-nm filaments, and can induce Alz50
immunoreactivity (which recognizes a disease-related epitope in tau) in vitro (32). However, there are no reports of
Ó 2007 American Association of Neuropathologists, Inc.
Transglutaminases in Neurodegenerative Disease
transglutaminase-catalyzed cross-linking of tau protein in
cells in culture. Transglutaminase may be capable of
stabilizing (i.e. Bspot-welding[) preformed tau oligomers
rather than inducing oligomer formation in cells in culture.
In vitro, transglutaminase 2 is also capable of crosslinking >-synuclein, a major component of Lewy bodies in
PD (21). The in vitro cross-linking of >-synuclein results in
the formation of high molecular weight polymers. Similarly,
in cells transfected with >-synuclein and transglutaminase 2,
Triton X-100-insoluble high molecular weight aggregates
are formed (21).
HD is an autosomal dominant neurodegenerative
disorder caused by an unstable CAG triplet repeat expansion in the open reading frame of the first exon coding for a
polyglutamine stretch in the amino terminus of the
huntingtin protein (34). Normal individuals have between
11 and 34 CAG repeats in the huntingtin gene. However,
patients with HD have 36 to 150 CAG repeats in the
huntingtin gene coding for an expanded polyglutamine
stretch (34Y36). In vitro and in cells in culture, huntingtin
protein with a expanded polyglutamine repeat region is an
excellent substrate for transglutaminase (8, 37, 38) whereby
the rate of the reaction increases over an order of magnitude
with 40 or more polyglutamine repeats (38). Furthermore,
transglutaminase catalyzes the formation of formic acid
soluble aggregates of mutant huntingtin protein in cells in
culture (29). However, in transfected cells, the predominant
form of huntingtin protein that contains the transglutaminasecatalyzed bond is a monomer (39).
TRANSGLUTAMINASE ISOENZYMES
To date, nine transglutaminase isoenzymes coded for
by distinct genes have been identified in mammals. Transglutaminase isoenzymes are differentially regulated and
show different substrate specificity (40). Transglutaminases
are found in a variety of mammalian tissues including the
central and peripheral nervous system. The most wellcharacterized isoenzymes expressed in human brain are
factor XIIIa, transglutaminase 2 (also known as tissue
transglutaminase), and transglutaminase 1 and 3 (7).
In rat brain, two alternatively spliced isoforms of
transglutaminase 2 have been described, a short and a long
form (40, 41). The mRNA expression of the two transglutaminase 2 isoforms in human brain has been reported in
AD cortex and in PSP brain but not in control cases (6, 42).
Interestingly, the short isoform of transglutaminase 2 mRNA
is not expressed in HD in a selectively vulnerable brain
region; rather, the long form of transglutaminase is increased
(39). A shorter human transglutaminase 2 isoform has also
been identified in human erythroleukemia cells. This shorter
human transglutaminase 2 is similar to the shorter rat
isoform in that the shorter isoform contains a different
carboxyl terminus than the longer isoform. Whereas the long
form is constitutively expressed, the short form is induced by
cytokines, such as interleukin-1A (41). Because cytokines,
including interleukin-1A, are upregulated in AD (43),
interleukin-1A could underlie the increased expression
of this short transglutaminase form in AD. There are
also reports of increased levels of mRNA coding for
259
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Muma
transglutaminase 1 and 2 in AD and PSP (6, 7). It is not
known whether there is an accompanying increase in protein
expression of transglutaminase 1 in AD (44) or an increase
in protein levels of the short isoform of transglutaminase 2
in either AD or PSP. There is an increase in transglutaminase 2 mRNA in substantia nigra of PD cases compared with
age-matched control cases (17), but the expression of the
short transglutaminase 2 isoform has not been examined.
Transglutaminase proteins are found in neurons in
brain regions with inclusion bodies and in the inclusion
bodies themselves. Transglutaminases are present in human
hippocampal neurons and in AD brain colocalize with
neurofibrillary tangles in hippocampal neurons (45). The
immunolabeling with transglutaminase antibodies was more
intense in AD hippocampus compared with age-matched
controls (45). Similarly, transglutaminase 2 has been
colocalized with mutant huntingtin in intranuclear inclusions
in HD brain (29). Transglutaminase 2 expression was
detected in neurons with Lewy bodies in PD substantia
nigra, but not in controls (17, 22).
TRANSGLUTAMINASE ACTIVITY
The activity of transglutaminase enzymes is highly
regulated. Transglutaminase activity is increased in pathologically vulnerable brain regions in the neurodegenerative
diseases thus far examined. In PSP, transglutaminase activity
is higher in the globus pallidus and pons, two selectively
vulnerable brain regions, compared with controls, whereas
activity in PSP occipital cortex, a neuropathologically spared
region, is comparable to control levels (6). In HD, neuronal
nuclei contain increased transglutaminase protein levels and
higher enzymatic activity compared with control cases
(8, 46). Similarly, the activity of transglutaminase is higher
in the cortex in AD cases compared with controls (7, 47).
REGULATION OF TRANSGLUTAMINASE
The regulation of transglutaminase has been examined
in several studies. The human transglutaminase 2 gene has
been analyzed: the gene contains 13 exons and 12 introns, and
exons 6 and 10 are alternatively spliced (48). Analysis of the
5 upstream region resulted in the identification of several
putative promoter sites including three potential SP1 sites
and two potential AP2 sites. Transglutaminase 2 also
functions in transmembrane signaling (49, 50). Transglutaminase transmits signals from >1-adrenoreceptors to phospholipase C-F1 in human heart. The two functions of
transglutaminase 2 are antagonistic: at high calcium concentrations the cross-linking function is active whereas at
low calcium concentrations GTP binding occurs. Similarly,
at high concentrations of GTP, the cross-linking function
is inhibited.
Calcium Regulates Transglutaminase Activity
Transglutaminase enzymes are calcium-dependent
zymogens containing a cysteine in their active site that is
unmasked only in the presence of calcium; thus, calcium is
their universal activator (11). A loss of calcium homeostasis
is a common characteristic of neurodegenerative diseases
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(51), and the regulation of intracellular calcium is altered
and levels are increased in aging (52).
Calmodulin Regulates Transglutaminase Activity
Calmodulin is a 17-kDa protein, which upon calcium
binding activates a host of enzymes (53). Calmodulin
activates transglutaminase in systems such as the human
erythrocyte cytoskeleton (54), human platelets, and chicken
gizzard (55). We observed colocalization of calmodulin with
huntingtin, transglutaminase 2, and the transglutaminasecatalyzed bonds within nuclei in HD cortex (56). Mutant
huntingtin associates with calmodulin as demonstrated by
affinity purification (57) and immunoprecipitation approaches
(56). The calmodulin inhibitor w5-hydrochloride prevents
cross-linking of huntingtin in cells that are cotransfected with
the amino terminal of huntingtin protein with an expanded
polyglutamine domain and transglutaminase 2 (56).
Transglutaminase Upregulation in Transgenic
Mouse Models of Neurodegenerative Diseases
The involvement of transglutaminase in transgenic
mouse models of neurodegenerative diseases has been
studied in models of HD and tauopathy but not PD or AD
(or more specifically in models of the amyloid pathology in
AD). Our data demonstrate that transglutaminase-catalyzed
cross-links are present in the neurofibrillary tangles and
paired helical filament tau from P301L tau transgenic mice
but not nontransgenic control mice and transglutaminase
activity is higher in the P301L tau transgenic mice (58).
The contribution of transglutaminase to the pathology
in HD mouse models has been studied more extensively.
Transglutaminase activity is higher in two HD transgenic
mouse models, R6/2 and YAC128 mice (59, 60). Transglutaminase 2 has been knocked out in two different transgenic mouse models of HD (61, 62). R6/1 and R6/2 HD
transgenic mice overexpress exon 1 of the human huntingtin
gene with an expanded CAG repeat (expressing 115 and 150
repeats, respectively) driven by the human huntingtin
promoter. These mice display age-related motor dysfunction,
weight loss, neuropathology, and a decreased life span
reminiscent of the human disease. Knockout of transglutaminase 2 in R6/1 and R6/2 HD transgenic mice increases
survival and prolongs motor function. Interestingly, ablation
of transglutaminase 2 increases aggregate formation in these
transgenic mice, suggesting that transglutaminase does not
contribute to the formation of inclusions in HD but
contributes to the pathophysiology by some other mechanism. Our results demonstrate that transglutaminase predominantly modifies monomeric huntingtin, suggesting that
transglutaminase catalyzes binding to a monamine or polyamine (39), consistent with the findings that transglutaminase does not increase huntingtin inclusions in HD transgenic
mice.
Cystamine is a transglutaminase inhibitor that has been
tested in HD transgenic mice (59, 60, 63). Cystamine reduced
transglutaminase activity, improved weight loss, reduced
motor dysfunction, and increased survival of the R6/2 HD
transgenic mice compared with saline-treated and untreated
littermates (60, 63). Treatment with cystamine starting at
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7 months of age in the YAC128 HD mouse model did not
improve motor dysfunction but did reduce transglutaminase
activity and reduced the loss of striatal volume and striatal
neurons (59). Neuropathologically, there was also a reduction in striatal atrophy in R6/2 mice treated early compared
with untreated and saline-treated mice (59, 60). Aggregates
of huntingtin protein were reduced 70% in the striatum and
50% in the cortex in R6/2 mice treated with cystamine
starting from 21 days of age, compared with untreated and
saline-treated mice (60). In R6/2 mice treated with cystamine starting at 7 weeks of age, there was no impact on the
number of intranuclear inclusions (63). One possibility is
that early treatment with cystamine is necessary to reduce
the intranuclear inclusions; however, results from the transglutaminase 2 knockouts in HD mice are not consistent with
this interpretation. Taking into account the results from the
studies using either transglutaminase 2 knockout or treatment with cystamine, a more likely explanation is that the
formation of inclusions is not dependent on transglutaminase. Furthermore, the results from these studies suggest that
loss of neurons in the striatum, motor dysfunction, and
longevity are not dependent on the formation of intracellular
inclusions. Transglutaminase may be inducing pathology via
some other mechanism such as the formation of smaller
oligomers of huntingtin protein or the stabilizing huntingtin
protein by the transglutaminase-catalyzed addition of a
mono- or polyamine. Indeed, in cells transfected with the
amino terminus of huntingtin protein with an expanded
polyglutamine domain, treatment with cystamine increased
cell survival and reduced monomeric huntingtin protein
containing the transglutaminase-catalyzed bond (39). Alternatively, cystamine increases expression of brain-derived
growth factor, which is neuroprotective (64). It has yet to be
determined whether knockout of transglutaminase 2 would
also increase expression of brain-derived growth factor.
Using the same dose and route of administration as in
the study of HD transgenic mice by Dedeoglu et al, (60),
treatment of P301L tau transgenic mice with cystamine did
not reduce transglutaminase activity in the spinal cord
(personal observation, 2006). Because we have previously
demonstrated that expression of the short splice variant of
transglutaminase 2 is increased in PSP (6) and not in HD (39),
perhaps cystamine does not inhibit the short splice variant.
Transglutaminase Upregulation With
Neuronal Injury
Neuronal injury, especially injury induced by excitotoxins or ischemia, has been associated with neurodegenerative disease processes. Transglutaminase is upregulated in
several models of neuronal injury. After a crush injury (i.e. an
axotomy) of rat superior cervical ganglion, transglutaminase activity is rapidly (within minutes) and transiently
increased in a calcium-dependent manner (65). Axotomy to
other peripheral nerves, such as the facial nucleus and
nodose ganglia, also results in a rapid increase in transglutaminase activity as well a second phase in which
transglutaminase activity is increased for numerous days
(66). Transglutaminase activity is also upregulated after
excitotoxic damage induced by glutamate exposure and after
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Transglutaminases in Neurodegenerative Disease
global cerebral ischemia (67). The neurotoxin, 1-methyl4-phenylpyridinium ion, induces parkinsonian-like pathology
and motor symptoms; exposure of neuronal cells to 1-methyl4-phenylpyridinium ion increases transglutaminase activity
(68).
DETECTION OF THE
TRANSGLUTAMINASE-CATALYZED BOND
Antibodies directed against the ?-(F-glutamyl) lysine
bonds (often abbreviated GGEL for Bgamma glutamyl
epsilon lysine[) such as 81D4 from Coval Laboratories
(Lyon, France) are a useful tool in research on neurodegenerative diseases and greatly facilitate detection of the
isopeptide bond over previous methods such as high pressure
liquid chromatography. Although the effectiveness of 81D4
on Western blots has been questioned, this antibody is
specific for the transglutaminase-catalyzed GGEL bond
when used for immunoprecipitation (18, 39) and ELISA
methods (19, 69). In vitro cross-linking of proteins and
cross-linking in transfected cells have demonstrated the
ability of this antibody to specifically and quantitatively
detect the transglutaminase-catalyzed GGEL bond.
ACTIONS OF TRANSGLUTAMINASE IN
NEURODEGENERATIVE DISEASES
Although mutations in tau can result in the formation
of tau filaments and neurodegeneration, the mechanisms
underlying the formation of tau filaments in AD are less
clear because tau mutations are not associated with AD.
Although the amyloid hypothesis is continually gaining
support from new studies, the connection between AA and
tau pathology remains tenuous. AA can increase enzymes
involved in the phosphorylation of tau, but tau phosphorylation is not sufficient to result in formation of tau filaments.
One of the mechanisms by which tau protein could be
perturbed in AD is via increases in cytokine activity and
intracellular levels of calcium caused by AA that could
increase transglutaminase expression and activity, respectively. The increase in transglutaminase activity and expression could then result in transglutaminase bonds in tau
monomers, oligomers, and filaments resulting in stabile
dysfunctional tau monomers and aggregation of stabilized
tau filaments. Cross-linked tau may not bind as well to
microtubules, resulting in an increase in free tau available
for polymer formation. Cross-links may make tau protein
resistant to degradation, leading to an increase in tau protein
available for filament formation. Alternatively, cross-linking
may be the mechanism by which tau assembles into
polymers as has been shown to occur in vitro (70). Lastly,
cross-links may stabilize tau polymers in neurofibrillary
tangles formed by some other mechanism.
Similarly, transglutaminase-catalyzed cross-linking
may lead to the stabilization of huntingtin protein monomers, oligomers, and small polymers. The transglutaminasemodified huntingtin protein is resistant to degradation (10)
and could thereby result in increased levels of abnormal
huntingtin protein in HD. Transglutaminase-catalyzed
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J Neuropathol Exp Neurol Volume 66, Number 4, April 2007
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cross-linking may be involved in the pathophysiology of
other CAG triplet repeat disorders as well (12, 34, 36).
Transglutaminase inhibitors could prevent stabilization of
monomers/oligomers and could be tested as possible treatments in these other CAG triplet repeat disorders. Further
studies are needed to determine the role(s) of transglutaminase in neurodegenerative diseases. Inhibitors of transglutaminase activity should be explored as potential therapeutic
strategies not only for HD and AD but also for a range of
neurodegenerative diseases with protein aggregates as a
pathologic feature.
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