Rat tau proteome consists of six tau isoforms: Implication for animal models of human tauopathies
Human brain encompasses six tau isoforms, containing either three (3R) or four (4R) repeat domains, all of which participate in the pathogenesis of human tauopathies. To investigate the role of tau protein in the disease, transgenic rat models have been created. However, unlike humans, it has been suggested that rat brain expresses only three 4R tau isoforms. Because of the significance of the number of tau isoforms for faithful reproducibility of neurofibrillary pathology in transgenic rat models, we reopened this issue. Surprisingly, our results showed that adult rat brain contains six tau isoforms like humans. Protein expression of 4R tau isoforms was ninefold higher than 3R isoforms. Furthermore, the protein levels of tau isoforms with none, one or two N-terminal inserts were 30%, 35%, and 35% of total tau, respectively. Moreover, amount and ratio of tau isoforms were developmentally regulated. The levels of 4R tau isoforms progressively increased from early postnatal period until adulthood, whereas the expression of 3R tau isoforms reached maximum at P10 and then gradually declined. Our results show that rat brain encompasses full tau proteome similar to humans. These findings support the use of rat as an animal model in human tauopathies research.
*Axon Neuroscience GmbH, Rennweg, Vienna, Austria
Institute of Neuroimmunology, AD Centre, Slovak Academy of Sciences, Dubravska cesta, Bratislava, Slovakia
àDepartment of Medical Biochemistry, Jessenius Faculty of Medicine, Comennius University, Martin, Slovak
Tau is one of the major brain microtubule-associated proteins
(MAP) found predominantly in axons. The protein tau
stabilizes and regulates dynamics of axonal microtubules
(Alonso et al. 1994; Iqbal et al. 1994). It is encoded by a
single gene whose mRNA is alternatively spliced to produce
six protein isoforms expressed in the adult human brain
(Goedert et al. 1989; Himmler et al. 1989). The tau isoforms
differ from each other by the presence of either three (3R) or
four (4R) C-terminal repeats and by the presence or absence
of one or two inserts in the N-terminal part of the molecule
(0N3R, 0N4R, 1N3R, 1N4R, 2N3R, 2N4R, see Fig. 1). The
3R and 4R in the C-terminal half of the protein have been
shown to bind microtubules (Evans et al. 2000). The 3R
isoforms bind to microtubules with lower avidity than the 4R
isoforms (Dayanandan et al. 1999). This difference seems to
play a role in the regulation of plasticity and process
formation and neurite elongation during neuronal develop-
ment (Shea et al. 1992; Goode and Feinstein 1994; Knowles
et al. 1994; Panda et al. 2003).
In Alzheimer’s disease (AD) and related neurodegenera-
tive tauopathies, all six tau protein isoforms undergoes
abnormal post-translational modiﬁcation that can inﬂuence
its conformational characteristics and thus gain a novel toxic
property (for review see (Buee et al. 2000; Iqbal and
Grundke-Iqbal 2006; Zilka et al. 2008). In several human
tauopathies altered 3R/4R-tau ratios have been observed
(Sergeant et al. 2005). The identiﬁcation of exonic and
intronic mutations in the tau gene in frontotemporal dementia
and parkinsonism linked to chromosome 17 established that
Received August 15, 2008; revised manuscript received December 9,
2008; accepted December 9, 2008.
Address correspondence and reprint requests to Michal Novak, MDV
DSc, Institute of Neuroimmunology, Slovak Academy of Sciences,
Dubravska cesta 9, Bratislava 84510, Slovakia.
Abbreviations used: 1-DE, one dimensional electrophoresis; 2-DE,
two dimensional electrophoresis; 3R tau, three-repeat tau; 4R tau, four-
repeat tau; AD, Alzheimer’s disease; IEF, isoelectric focusing; LMR, low
molecular weight; MAP, microtubule-associated protein; Mr, molecular
mass; P10, postnatal day 10; PHF, paired helical ﬁlaments; pI, isoelectric
point; RT, room temperature; SDS–PAGE, sodium dodecyl sulfate–
polyacrylamide gel electrophoresis; TBST, Tris-buffered saline con-
taining Tween 20.
Human brain encompasses six tau isoforms, containing either
three (3R) or four (4R) repeat domains, all of which participate
in the pathogenesis of human tauopathies. To investigate the
role of tau protein in the disease, transgenic rat models have
been created. However, unlike humans, it has been sug-
gested that rat brain expresses only three 4R tau isoforms.
Because of the signiﬁcance of the number of tau isoforms for
faithful reproducibility of neuroﬁbrillary pathology in transgenic
rat models, we reopened this issue. Surprisingly, our results
showed that adult rat brain contains six tau isoforms like hu-
mans. Protein expression of 4R tau isoforms was ninefold
higher than 3R isoforms. Furthermore, the protein levels of
tau isoforms with none, one or two N-terminal inserts were
30%, 35%, and 35% of total tau, respectively. Moreover,
amount and ratio of tau isoforms were developmentally reg-
ulated. The levels of 4R tau isoforms progressively increased
from early postnatal period until adulthood, whereas the
expression of 3R tau isoforms reached maximum at P10 and
then gradually declined. Our results show that rat brain
encompasses full tau proteome similar to humans. These
ﬁndings support the use of rat as an animal model in human
Keywords: animal model, brain, neurodegeneration, rat tau
proteome, tau isoforms, tauopathies.
J. Neurochem. (2009) 108, 1167–1176.
JOURNAL OF NEUROCHEMISTRY | 2009 | 108 | 1167–1176 doi: 10.1111/j.1471-4159.2009.05869.x
2009 The Authors
Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1167–1176 1167
dysfunction of tau alone can induce and drive degeneration
of neuronal and glial cells and can directly lead to dementia
(Hutton et al. 1998; Gotz et al. 2007). Over the past decade,
the research has moved to the development of genetically
modiﬁed rodents reproducing key clinical, histopathological
and molecular hallmarks of human tauopathies by expression
of wild type, mutated or structurally modiﬁed tau proteins.
However, it has been shown that adult rodent brain may not
express full set of tau protein isoforms that are found in the
human brain. Most authors suggested that adult rat brain
expressed only three 4R tau isoforms at the protein level
(Janke et al. 1999; Chambers et al. 2000; Takuma et al.
2003) as well as at the mRNA level (Kosik et al. 1989;
Ferreira et al. 1997; Janke et al. 1999; Chambers et al.
2000), while few authors hypothesized that rat brain may
contain more than four tau isoforms (Tatebayashi et al. 1999;
Gorath et al. 2001).
The aim of this study was to clarify complete tau proteome
in the rat brain to justify the use of rat as an animal model in
human tauopathy research. We solved the issue of number of
tau isoforms present in the adult rat brain and identiﬁed their
distribution patterns in different brain areas. Furthermore, we
characterized the ontogenesis of the rat tau proteome during
the postnatal brain development.
Material and methods
The spontaneously hypertensive rat (SHR) strain used in this study
was derived from a progenitor strain of SHR (SHR/Ola) that
descends from inbred SHR originally obtained from the National
Institutes of Health. This progenitor strain of SHR has been
maintained by brother · sister mating at the Czech Academy of
Sciences (Prague, Czech Republic) for 15 yr. The rats were in the
F48 generation when the SHR colony was established in Prague.
The development of hypertension in SHR rats occurs at 8 weeks of
age. In the adult rats, the blood systolic pressure ranged from 180 to
190 mmHg and the diastolic blood pressure is about 125 mmHg
(Kren et al. 1997).
All animals were housed under standard laboratory conditions
and were kept under diurnal lighting regime (12 h light/dark cycles
with light starting at 7:00
). All experiments on animals were
carried out according to the institutional animal care guidelines
conforming to international standards and were approved by The
State Veterinary and The Food Committee of the Slovak Republic
and by the Ethics Committee of Institute of Neuroimmunology,
Slovak Academy of Sciences, Bratislava. Efforts were made to
minimize the number of animals utilized and to limit discomfort,
pain, or any other suffering of the experimental animals used.
The following monoclonal antibodies were used in this study: DC25
recognizes tau residues 347–354 (culture supernatant diluted 1 : 10;
Axon Neuroscience, Vienna, Austria); RD3, speciﬁc for 3R tau
isoforms (dilution 1 : 1000; Millipore, Billerica, MA, USA); and
DC4R, speciﬁc for 4R tau isoforms (culture supernatant diluted
1 : 1; Axon Neuroscience).
The brain tissues were homogenized in ice-cold extraction buffer
(20 mM Tris–HCl, pH 8, 100 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 0.5% Triton X-100, 20 mM NaF, and 1 mM activated
) supplemented with protease inhibitor cocktail (Complete
without EDTA; Roche, Manheim, Germany) using the Omni TH
tissue homogenizator (OMNI H; Omnilab, Bremen, Germany). The
homogenates were centrifuged at 20 800 g for 20 min at 4C and
the supernatants were transferred into fresh tubes. Concentration of
total proteins was determined by Bio-Rad Protein Assay (Bio-Rad
Laboratories, Hercules, CA, USA).
The samples were dialyzed in slide-A-Lyzer Mini Dialysis Units
(Pierce, Rockford, IL, USA) at 4C for 6 h in 100 mM Tris, pH 8,
and 1 mM phenylmethylsulfonyl ﬂuoride. The buffer was changed
every 2 h. After dialysis, the samples were supplemented with b-
mercaptoethanol (5 lL/mL) and heated at 95C for 5 min. The
boiled samples were cooled down on ice and precipitated proteins
removed by centrifugation at 20 800 g for 20 min at 4C. The
clariﬁed supernatants were supplemented with alkaline phosphatase
(Sigma-Aldrich, St Louis, MO, USA) to the ﬁnal concentration of
20 U/mL. The samples were dephosphorylated overnight at 37C.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–
PAGE) was performed according to Laemmli (1970). The superna-
tants were mixed with equal volume of 2x SDS sample loading
buffer and heated at 95C for 5 min. Proteins were separated in 12%
Isoelectric focusing (IEF) was performed in Protean IEF Cell (Bio-
Rad Laboratories) using immobilized pH gradient gel strips
according to manufacturer’s recommendations (manual ‘2-D Elec-
trophoresis for Proteomics’). Brieﬂy, the brain extracts were mixed
with ice-cold 96% ethanol (1 : 5), the proteins were precipitated at
)20C for 2 h and then collected by centrifugation at 20 800 g for
30 min at 4C. The pellets were washed once with ice-cold 96%
ethanol, dried at 25C) for 1 min, and dissolved in thiourea/urea
buffer: 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimeth-
Fig. 1 Human tau isoforms. The human brain expresses six tau iso-
forms. They differ by the absence or presence of one or two 29 amino
acids inserts (N1 and N2) in the amino-terminal part, and by the
presence of three (R1, R3, and R4) or four (R1, R2, R3, and R4)
repeat-regions in the carboxy-terminal part.
Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1167–1176
2009 The Authors
1168 | J. Hanes
ylammonio]-1-propanesulfonate, 0.5% ampholytes 3–11, and 1.2%
Destreak reagent (both GE Healthcare, Piscataway, NJ, USA). The
strips (Immobiline Dry Strip gel, pH 3–11 non-linear, 7 cm; GE
Healthcare) were rehydrated with the samples (for native samples
150 lg of protein was used; for the dephosphorylated samples its
equivalent prepared from 150 lg of protein) dissolved in thiourea/
urea buffer at 20C for 14 h. IEF was performed at 20C using the
following program: 2 h at 300 V, 30 min linear gradient to 1000 V,
2 h linear gradient to 5000 V, and constant 5000 V until 10 000 V h
were reached, whereby maximal current per one strip was limited to
50 lA. If necessary, the focused strips were frozen at )70C. The
second-dimension was carried out on 12% SDS–PAGE gels at
constant 15 mA per gel.
After the electrophoresis, the separated proteins were transferred to
0.2 lm Protran nitrocellulose membrane (Whatman, Maidstone,
UK) in 10 mM N-cyclohexyl-3-aminopropanesulfonic acid, pH 11,
in Mini Trans-Blot Cell (Bio-Rad Laboratories). The membranes
were blocked for 30 min in 5% (w/v) milk in Tris-buffered saline
containing Tween 20 (TBST; 20 mM Tris–HCl, pH 7.4, 0.15 M
NaCl, and 0.1% Tween 20), then incubated with primary antibody
diluted in 5% (w/v) milk in TBST for 1 h at 25C, washed four
times for 4 min in TBST, followed by incubation with goat anti
mouse secondary antibody conjugated to horseradish peroxidase
diluted in 5% (w/v) milk in TBST (dilution 1 : 4000; DAKO,
Glostrup, Denmark) for 30 min at 25C and washed four times with
TBST for 4 min. Blots were developed with Supersignal West Pico
Chemiluminiscent Substrate (Pierce) and visualized by Lumines-
cence reader LAS 3000 (FUJI Photo Film, Tokyo, Japan).
Quantitative analysis of tau isoforms
Dephosphorylated brain samples were separated by SDS–PAGE
together with serial dilution of recombinant human tau proteins
2N4R (2.5, 5, 10, 15, and 25 ng/well) and 2N3R (0.5, 1, 2, 3, 4, and
5 ng/well) for quantiﬁcation of rat 4R and 3R tau isoforms,
respectively (data not shown). Blots were developed with Super-
signal West Pico Chemiluminiscent Substrate and visualized by
Luminescence reader LAS 3000. The band intensities were
quantiﬁed using Advanced Image Data Analyzer (Aida) software
(Raytest, Straubenhardt, Germany).
Rat tau proteome encompasses six tau isoforms similar to
To analyze the number of tau isoforms in the rat brain we
used two speciﬁc monoclonal antibodies RD3 and DC4R,
which speciﬁcally recognize either 3R or 4R tau proteins,
respectively, and pan-tau antibody DC25 recognizing all tau
proteins. Dephosphorylated tau proteins from adult rat brain
were separated into six protein groups with different apparent
molecular weights (Fig. 2a, total tau). Three of them were
recognized speciﬁcally with RD3 antibody (Fig. 2a, three-
repeat tau) and another three with DC4R antibody (Fig. 2a,
four-repeat tau). As a positive control we used the human
parietal cortex, which was extracted and analyzed in a similar
way like the rat brain samples. Six tau isoforms were
detected using the pan-tau antibody, whereby three of them
were recognized as 3R tau proteins and another three as 4R
tau proteins (Fig. 2b). Comparison of rat and human tau
proteomes revealed that their two-dimensional electrophore-
sis (2-DE) patterns were very similar. In human and rat brain,
tau isoforms without N-terminal inserts appeared as a single
spot with isoelectric point (pI) 8.5. All tau isoforms with one
or two N-terminal insert appeared as rows of several spots
with the same molecular mass (Mr) but with different pI. The
rat isoforms were composed of the following number of spots
(with pI): 1N3R, four spots (pI 7–8); 1N4R, four spots (pI
6.8–8.2); 2N3R, three spots (pI 6.3–6.8); and 2N4R, seven
spots (pI 6.5–7.5). The composition of human isoforms was
the following: 1N3R, six spots (pI 6.8–8); 1N4R, seven spots
(pI 6.9–8.1); 2N3R, ﬁve spots (pI 6–7), and 2N4R, three
spots (pI 6.7–7.1).
The rat tau proteome differs between the individual rat
After deﬁning the tau proteome, we compared the expression
levels and composition of individual tau isoforms in three
brain regions of adult rat: brainstem, hippocampus, and
cortex. Native brain extracts were subjected to one-dimen-
sional electrophoresis (1-DE) immunoblot analysis using 3R
tau and 4R tau speciﬁc antibodies. Tau proteome was
separated into 18 protein bands of which half were 3R tau
and another half were 4R tau species. The 4R tau isoforms
showed very similar pattern in all three brain tissues.
However, the total amount of 4R tau isoforms was signif-
icantly lower in the brainstem compared with the hippocam-
pus and cortex. It is noteworthy that both the pattern and the
amount of rat 3R isoforms differed between brain areas. Two
groups of 3R tau species were observed, one with Mr 57–
63 kDa (high molecular weight 3R tau) and the second with
Mr 48–53 kDa (low molecular weight; LMR 3R tau). The
LMR 3R tau showed similar pattern in all studied brain areas,
while the high molecular weight 3R tau was much more
abundant in the brainstem compared with the hippocampus
and cortex (Fig. 3a).
To identify the individual tau isoforms we dephosphoryl-
ated brain samples and subjected them to the immunoblot
analysis. The analysis revealed that tau proteome in all three
studied tissues encompassed all six tau isoforms, however,
their expression pattern was not uniform. The tau expression
patterns in the hippocampus and cortex were very similar and
differed markedly from the pattern of tau proteins in the
brainstem. Expression levels of all 4R tau proteins and the
0N3R tau isoform in the hippocampus and cortex were
higher compared with the brainstem. On the contrary, protein
expression of 1N3R tau and 2N3R tau appeared higher in
brainstem. Interestingly, in all three rat brain areas tau
2009 The Authors
Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1167–1176
Rat tau proteome |
isoforms with none, one or two N-terminal inserts were
equally abundant and their expression levels were about
30%, 35%, and 35% of total tau, respectively. Moreover, we
found that the 4R/3R ratio of tau isoforms differed between
the brain areas; 9 : 1 in the cortex, 7 : 1 in the hippocampus,
and 6 : 1 in the brainstem (Fig. 3b).
The tau proteome from the three brain areas was also
subjected to 2-DE immunoblot analysis. In comparison to the
1-DE results, we additionally found that the 4R tau isoforms
with N-terminal inserts differed between the brain areas.
Number of spots in these two isoforms was reduced in the
brainstem when compared with other brain areas. Interest-
ingly, 3R tau isoforms with N-terminal inserts composed of
almost the same numbers of spots in all three studied brain
regions, but the expression levels were signiﬁcantly different.
Furthermore, tau isoforms without N-terminal inserts (0N3R
and 0N4R) appeared as a single spot in all three brain areas
The expression of the six rat tau isoforms is
To reveal the expression pattern of rat tau proteome during
postnatal brain development, we analyzed brain extracts
from 5-, 10-, 20-, 30-, and 3-month-old animals. We found
that on the postnatal day 5 (P5), exclusively the expression
of the 0N3R isoform was detected. Expression of the 0N3R
isoform increased during the ﬁrst days of the postnatal
period, reached maximum on P10, and then gradually
decreased with the age of the animal. On the P10, all
additional ﬁve tau protein isoforms appeared, but their
expression was very low. Expression of all 4R tau isoforms
increased from P10 until adulthood. Amount of 1N3R tau
protein isoforms was the highest on P20 and gradually
decreased until adulthood of rat. The 2N3R tau isoform
reached maximal expression level on P30, which was similar
to that in adulthood. Expression of N-terminal inserts
containing 3R tau isoforms (1N3R and 2N3R) was much
lower compared with N-terminal inserts containing 4R
isoforms (1N4R and 2N4R). This difference was observed
during whole brain development and was the highest in adult
animals. Comparison of 3R and 4R tau proteins showed that
expression levels of 4R tau proteins progressively increased
from early postnatal development (P5) until adulthood,
whereas 3R tau proteins reached maximum at P10 and then
gradually decreased reaching lowest level in adult animals
(Fig. 5a and b).
Fig. 2 Rat tau proteome encompasses six tau isoforms similar to
humans. Dephosphorylated samples from adult rat cortex (3-month-
old rat) (a) and human parietal cortex (b) were separated by 2-DE. The
3R tau isoforms (three-repeat tau), 4R tau isoforms (four-repeat tau),
and all tau proteins (total tau) were detected with RD3 antibody, DC4R
antibody and DC25 antibody, respectively. All six tau isoforms were
detected in both human cortex and rat brain (arrowheads). It is note-
worthy that human and rat tau proteome patterns are very similar. Tau
isoforms without N-terminal inserts appeared as a single spot in both
human and rat in contrast to all tau isoforms with one or two N-terminal
insert which appeared as rows of several spots. The positions of
molecular weight marker (kDa) and isoelectric point marker (pI) are
shown on the left and bellow, respectively.
Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1167–1176
2009 The Authors
1170 | J. Hanes
Moreover, the 4R/3R ratio of tau isoforms changed during
brain development from 1 : 4 on P10 to 9 : 1 in adult
animals. Further, the protein levels of tau isoforms with none,
one or two N-terminal inserts changed from 90%, 7%, and
3% of total tau (P10) to 30%, 36%, and 33% in adult
animals, respectively (Fig. 5c).
Human MAP tau is coded by a single gene located on
chromosome 17q21. Tau expression is developmentally
regulated, only the shortest tau isoform is expressed in the
fetal brain. During the postnatal period, alternative splicing of
the tau gene generates six isoforms of the tau protein. The
isoforms differ from each other by the presence of either 3R or
4R tau in the carboxy-terminal, and the presence or absence of
one or two inserts of 29 amino acids in the amino-terminal
region (Buee et al. 2000). The C-terminal repeat-region binds
to microtubules and the N-terminal region interacts with the
plasma membrane and other cytoskeletal proteins (Goedert
and Jakes 1990; Gustke et al. 1994; Brandt et al. 1995).
Tau becomes functionally and structurally altered in
several neurodegenerative tauopathies including AD. Re-
cently, it has been shown that abnormal phosphorylation of
tau is able to inhibit the microtubule assembly, to disrupt the
microtubule system, to self-aggregate into ﬁlamentous
structures, and to induce self-assembly of all six tau isoforms
(Alonso et al. 1996, 1997, 2001a,b). Tau truncation has been
proposed as an early and important step in the development
of neuroﬁbrillary lesions (Novak et al. 1991, 1993). More-
over, we demonstrated that human truncated tau protein
promoted abnormal microtubule assembly in vitro and
induced formation of neuroﬁbrillary tangles in the transgenic
rat model for tauopathy (Zilka et al. 2006).
The aggregation of disease modiﬁed tau protein into paired
helical ﬁlaments (PHFs), the major constituents of neuroﬁ-
brillary tangles, is a prominent pathological feature in AD
and related tauopathies (Grundke-Iqbal et al. 1986a,b;
Wischik et al. 1988a,b; Novak et al. 1991, 1993; Goedert
et al. 1992; Novak 1994). Interestingly, PHFs from AD brain
consist of three major tau bands of 60, 64, and 68 kDa
referred to as A68 or PHF-tau (Lee et al. 1991). Analysis of
dephosphorylated A68 showed that it was composed of all
six tau isoforms, each in a hyperphosphorylated state
(Goedert et al. 1992).
In recent years, several tau transgenic rodent models have
been developed that manifest phenotypic features similar to
AD and related tauopathies. It has been found that rodents
unlike humans encompass exclusively 4R tau isoforms (Gotz
2001; Mandelkow et al. 2007; Sennvik et al. 2007). So far
almost all papers describing rat tau proteome reported only
up to four tau isoforms: 0N3R, 0N4R, 1N4R and 2N4R
(Kosik et al. 1989; Chambers and Muma 1997; Janke et al.
1999; Takuma et al. 2003). Only, very few papers hypoth-
Fig. 3 The rat tau proteome differs between rat brain areas. (a)
Native tau proteome was separated in 18 protein bands, nine of
them were 3R tau speciﬁc, and nine of them 4R tau speciﬁc. The
amount of 4R tau isoforms in brainstem appeared signiﬁcantly lower
compared with hippocampus and cortex. In hippocampus and cortex,
higher molecular mass species of 3R tau proteins were almost
missing (arrowheads). (b) Analysis of dephosphorylated rat brain
samples revealed that tau proteomes from brainstem, hippocampus,
and cortex consisted of all six tau protein isoforms. Interestingly,
brainstem proteome markedly differed from those of hippocampus
and cortex. Most prominent difference was observed in expression
levels of 2N3R and 1N3R tau isoforms which were more abundant in
brainstem compared with other two studied brain areas. (c) Distri-
bution of tau isoforms within rat brainstem, hippocampus, and cortex.
Highest expression levels of 4R tau proteins were observed in cortex
followed by hippocampus and lowest in brainstem. Similar expression
pattern was observed also for 0N3R tau isoform. In contrast to them
2N3R and 1N3R abundance was the highest in brainstem followed by
hippocampus and lowest in cortex. (d) Quantitative analysis of the rat
tau proteome in three different brain areas. Expression levels of 4R
tau proteins compared with 3R tau isoforms were ninefold higher in
cortex, sevenfold higher in hippocampus, and sixfold higher in
brainstem. It is noteworthy that expression levels of tau isoforms with
none, one, or two N-terminal inserts were equally abundant. 3R,3R
tau proteins; 4R, 4R tau proteins; 3R + 4R, sum of 3R and 4R tau
proteins; 2N, 1N, and 0N, tau isoforms with 2, 1, and none N-terminal
inserts; all, expression level of all 3R or 4R tau isoforms in particular
2009 The Authors
Rat tau proteome |
esized on the presence of more than four tau isoforms in the
rat brain (Mawal-Dewan et al. 1994; Tatebayashi et al. 1999;
Gorath et al. 2001).
The composition of rat tau proteome could play crucial role
in the development of neuroﬁbrillary degeneration in the rat
models of human tauopathies. Therefore, we reopened the
issue of the number of tau isoforms in the rat brain. In our study,
we applied 1-DE and 2-DE to analyze the composition of rat
tau proteome. To distinguish individual tau isoforms, we used
monoclonal antibodies recognizing speciﬁcally only 3R tau
isoforms (RD3), 4R tau proteins (DC4R), or all six isoforms
(DC25). Resulting analysis revealed six tau protein groups
with different molecular weights each of them containing one
to six protein spots possessing different pI. While tau isoforms
containing N-terminal inserts (1N3R, 2N3R, 1N4R, and
2N4R) consisted of several protein spots (three to six), both
tau isoforms without N-terminal inserts (0N3R and 0N4R)
appeared as a single spot. The appearance of several protein
spots (having the same molecular weight but different pI) in tau
proteins containing N-terminal inserts can be because of either
presence of other post-translational modiﬁcations than phos-
phorylation, incomplete dephosphorylation of tau proteins, or
combinations of both of these possibilities. Based on this
observation, it can be expected that N-terminal inserts of tau
protein may have different function and contain partial
secondary structure compared with other parts of tau protein.
It was already suggested that they could contain some
regulatory elements (Hirokawa et al. 1988).
Furthermore, we found that tau proteome is differently
expressed in various brain areas. In the hippocampus and
cortex, tau proteome revealed almost identical molecular
pattern that signiﬁcantly differed from the brainstem. Cortex
and hippocampus expressed higher amount of three 4R tau
isoforms and the shortest 3R tau isoform compared with
brainstem. On the other side, tau proteome in brainstem
contained higher amount of 3R tau isoforms possessing N-
terminal inserts. Quantiﬁcation of dephosphorylated 3R tau
isoforms with two, one, or zero N-terminal inserts and the
corresponding 4R tau isoforms from three different brain
areas showed that the ratio of 4R tau to 3R tau isoforms (4R/
3R) was about 9 : 1. Further, we found that 0N, 1N, and 2N
tau isoforms composed about 30%, 35%, and 35% of total
tau, respectively. In contrast to rat in adult human brain, the
ratio of 3R tau to 4R tau isoforms is 1 : 1 (Goedert and
Jakes 1990) and the 0N, 1N, and 2N tau isoforms comprise
about 37%, 54%, and 9% of total tau, respectively (Hong
Fig. 4 Two-DE analysis of rat proteome in brainstem, hippocampus,
and cortex. The 3R tau isoforms (three-repeat tau) and 4R tau iso-
forms (four-repeat tau) were detected with RD3 and by DC4R anti-
body, respectively. In all three studied brain areas, tau isoforms
without N-terminal inserts (0N3R and 0N4R) appeared as a single
spot. The one or two N-terminal insert containing tau isoforms ap-
peared as rows of several spots with same Mr and different pIs. Spots
present in the 2N4R and 1N4R isoforms showed different distribution
of their abundance. High abundant were identiﬁed middle spots in
2N4R versus spots with most basic pIs in 1N4R isoform to 1N4R tau
protein where the high abundant appeared species with the most basic
pI (four-repeat tau). 3R tau proteins within each studied brain area
showed similar representation. Six tau isoforms detected in all three
rat brain tissues are marked by arrowheads. Molecular weight marker
(kDa) and isoelectric point marker (pI) are shown on left and bellow,
2009 The Authors
1172 | J. Hanes
Fig. 5 The expression of the six rat tau
isoforms is developmentally regulated. (a)
The 3R tau isoforms (three-repeat tau), 4R
tau isoforms (four-repeat tau), and all tau
proteins (total tau) were detected with RD3,
DC4R, and DC25 antibodies, respectively,
in developing and adult rat brains. All six tau
protein isoforms were detected already on
P10. Arrowheads with solid and dashed
lines point to 3R tau isoforms and 4R tau
isoforms, respectively. The positions of
molecular weight marker (kDa) and iso-
electric point marker are shown on the left
and bellow, respectively. (b) Distribution of
tau isoforms in rat brain during postnatal
development. Expression levels of 4R tau
isoforms gradually increased from P10 until
adulthood whereas 3R tau isoforms
reached maximum on P10 and then grad-
ually decreased. The highest expression
level of 1N3R isoform appeared on P20 and
then decreased; the 2N3R was most
abundant on P30 and in adulthood. (c)
Quantitative analysis of expression levels of
six tau isoforms during postnatal develop-
ment. The 4R/3R ratio of tau isoforms
changed during brain development from
1 : 4 on P10 to 9 : 1 in adult animals. The
expression levels of tau isoforms with one
and two N-terminal inserts gradually in-
creased from 5% of total tau proteins on
P10 to 35% in adult animals. 3R, 3R tau
proteins; 4R, 4R tau proteins; 3R + 4R,
sum of 3R and 4R tau proteins; 2N, 1N and
0N, tau isoforms with 2, 1, and none N-
terminal inserts; all, expression level of all
3R or 4R tau isoforms in particular brain
2009 The Authors
Rat tau proteome |
et al. 1998). These ﬁnding showed that rat tau proteome
contains lower levels of 3R tau species but the higher amount
of tau isoforms with two N-terminal inserts when compared
with human brain. Previously, it has been shown that 4R tau
isoforms interact more efﬁciently with microtubules than 3R
tau isoforms (Goedert and Jakes 1990; Gustke et al. 1994). It
is plausible that the loss of 4R tau predominance and
quantitative decrease of tau isoforms with two inserts during
evolution could be linked to the complexity of nerve
interconnections. More sophisticated synaptic networks
require greater degrees of intracellular regulation. Thus,
composition of human tau proteome may represent phylo-
genetic advance in the human brain.
Previously, it has been shown that the expression of rat tau
is developmentally regulated (Mawal-Dewan et al. 1994;
Takuma et al. 2003). However, until recently, studies on the
tau ontogenetic pattern were mainly focused on the devel-
opmental changes in the amino terminal region or develop-
mental regulation of the tau phosphorylation state, while no
attention was paid to the isoform expression pattern during
early postnatal period. Therefore, we analyzed developmen-
tal regulation of all tau isoforms in the rat brain. We found
that the expression of fetal tau isoform (0N3R) increased
during ﬁrst days of rat’s life, reached maximum on P10 and
then continuously decreased within the age of animals. These
ﬁndings are consistent with previous developmental studies
(Kosik et al. 1989; Mawal-Dewan et al. 1994; Ferreira et al.
1997). Interestingly, on P10, a time that corresponds to active
synapse formation (Ferreira et al. 1997), we already detected
all ﬁve adult tau isoforms (0N4R, 1N3R, 1N4R, 2N3R, and
2N4R). While the protein levels of 4R tau isoforms
progressively increased from early postnatal development
until adulthood, the expression of 3R tau isoforms reached
maximum at P10 and then gradually decreased. Similarly to
tau protein, 3R and 4R forms of the LMR MAP2 variants
show different patterns of developmental regulation, which
are reﬂected in the levels of both their protein and mRNAs in
postnatal rat brain. While levels of the 3R MAP2 are high at
early stages and then decline, the 4R isoforms appears late in
development and are expressed exclusively in adult brain
(Doll et al. 1993; Ferhat et al. 1998). We suggest that the late
appearance of 4R tau isoforms in the brain postnatal
development may play signiﬁcant role in the stabilization
of neuronal circuitry during this period and thus may
contribute to the age-restricted brain plasticity.
Several independent immunocytochemical studies showed
that tau localization was conﬁned mainly to the axonal ﬁbers
and partially to the neuronal somatodendritic compartment.
Tau neuronal distribution was well characterized in the rat
cortex, hippocampus, cerebellum, brainstem, and spinal cord
(Binder et al. 1985; Schwab et al. 1994; Gartner et al. 1998).
On the contrast, other studies reported that tau immunore-
activity is not restricted to neurons but was found also in the
oligodendroglial and astroglial cells (Migheli et al. 1988;
Straiko et al. 2007). However, glial tau represents only minor
fraction of rat proteome. On the other side, it remains to be
investigated whether 3R and 4R tau isoforms are differen-
tially distributed in neuronal and glial cells in the rat brain.
In conclusion, the spatial and temporal study of rat tau
proteome revealed that rat brain encompasses six tau
isoforms similar to human. These ﬁndings further substan-
tiate the use of rat for the study of molecular mechanisms of
the tau-driven neuroﬁbrillary degeneration in human tauo-
pathies including AD.
This work was supported by Axon Neuroscience and research grants
VEGA 2/7129/27, 2/6183/26, APVV 0603-06, APVV 0631-07,
APVV 0559-07, LPP-0326-06, and LPP-0363-06.
Alonso A. C., Zaidi T., Grundke-Iqbal I. and Iqbal K. (1994) Role of
abnormally phosphorylated tau in the breakdown of microtubules
in Alzheimer disease. Proc. Natl Acad. Sci. USA 91 , 5562–5566.
Alonso A. C., Grundke-Iqbal I. and Iqbal K. (1996) Alzheimer’s
disease hyperphosphorylated tau sequesters normal tau into tan-
gles of ﬁlaments and disassembles microtubules. Nat. Med. 2, 783–
Alonso A. D., Grundke-Iqbal I., Barra H. S. and Iqbal K. (1997)
Abnormal phosphorylation of tau and the mechanism of Alzheimer
neuroﬁbrillary degeneration: sequestration of microtubule-associ-
ated proteins 1 and 2 and the disassembly of microtubules by the
abnormal tau. Proc. Natl Acad. Sci. USA 94, 298–303.
Alonso A., Zaidi T., Novak M., Grundke-Iqbal I. and Iqbal K. (2001a)
Hyperphosphorylation induces self-assembly of tau into tangles of
paired helical ﬁlaments/straight ﬁlaments. Proc. Natl Acad. Sci.
USA 98, 6923–6928.
Alonso A. D., Zaidi T., Novak M., Barra H. S., Grundke-Iqbal I. and
Iqbal K. (2001b) Interaction of tau isoforms with Alzheimer’s
disease abnormally hyperphosphorylated tau and in vitro phos-
phorylation into the disease-like protein. J. Biol. Chem. 276,
Binder L. I., Frankfurter A. and Rebhun L. I. (1985) The distribution of
tau in the mammalian central nervous system. J. Cell Biol. 101,
Brandt R., Leger J. and Lee G. (1995) Interaction of tau with the neural
plasma membrane mediated by tau’s amino-terminal projection
domain. J. Cell Biol. 131, 1327–1340.
Buee L., Bussiere T., Buee-Scherrer V., Delacourte A. and Hof P. R.
(2000) Tau protein isoforms, phosphorylation and role in neuro-
degenerative disorders. Brain Res. Brain Res. Rev. 33, 95–130.
Chambers C. B. and Muma N. A. (1997) Tau mRNA isoforms following
sciatic nerve axotomy with and without regeneration. Brain Res.
Mol. Brain Res. 48, 115–124.
Chambers C. B., Sigurdsson E. M., Hejna M. J., Lorens S. A., Lee J. M.
and Muma N. A. (2000) Amyloid-beta injection in rat amygdala
alters tau protein but not mRNA expression. Exp. Neurol. 162,
Dayanandan R., Van Slegtenhorst M., Mack T. G., Ko L., Yen S. H.,
Leroy K., Brion J. P., Anderton B. H., Hutton M. and Lovestone S.
(1999) Mutations in tau reduce its microtubule binding properties
in intact cells and affect its phosphorylation. FEBS Lett. 446, 228–
2009 The Authors
1174 | J. Hanes
Doll T., Meichsner M., Riederer B. M., Honegger P. and Matus A.
(1993) An isoform of microtubule-associated protein 2 (MAP2)
containing four repeats of the tubulin-binding motif. J. Cell Sci.
106(Pt. 2), 633–639.
Evans D. B., Rank K. B., Bhattacharya K., Thomsen D. R., Gurney M.
E. and Sharma S. K. (2000) Tau phosphorylation at serine 396 and
serine 404 by human recombinant tau protein kinase II inhibits
tau’s ability to promote microtubule assembly. J. Biol. Chem. 275,
Ferhat L., Represa A., Ferhat W., Ben-Ari Y. and Khrestchatisky M.
(1998) MAP2d mRNA is expressed in identiﬁed neuronal popu-
lations in the developing and adult rat brain and its subcellular
distribution differs from that of MAP2b in hippocampal neurones.
Eur. J. Neurosci. 10, 161–171.
Ferreira A., Lu Q., Orecchio L. and Kosik K. S. (1997) Selective
phosphorylation of adult tau isoforms in mature hippocampal
neurons exposed to ﬁbrillar A beta. Mol. Cell. Neurosci. 9, 220–
Gartner U., Janke C., Holzer M., Vanmechelen E. and Arendt T. (1998)
Postmortem changes in the phosphorylation state of tau-protein in
the rat brain. Neurobiol. Aging 19, 535–543.
Goedert M. and Jakes R. (1990) Expression of separate isoforms of
human tau protein: correlation with the tau pattern in brain and
effects on tubulin polymerization. EMBO J. 9, 4225–4230.
Goedert M., Spillantini M. G., Jakes R., Rutherford D. and Crowther
R. A. (1989) Multiple isoforms of human microtubule-associated
protein tau: sequences and localization in neuroﬁbrillary tangles of
Alzheimer’s disease. Neuron 3, 519–526.
Goedert M., Spillantini M. G., Cairns N. J. and Crowther R. A. (1992)
Tau proteins of Alzheimer paired helical ﬁlaments: abnormal
phosphorylation of all six brain isoforms. Neuron 8, 159–168.
Goode B. L. and Feinstein S. C. (1994) Identiﬁcation of a novel
microtubule binding and assembly domain in the developmen-
tally regulated inter-repeat region of tau. J. Cell Biol. 124, 769–
Gorath M., Stahnke T., Mronga T., Goldbaum O. and Richter-Landsberg
C. (2001) Developmental changes of tau protein and mRNA in
cultured rat brain oligodendrocytes. Glia 36, 89–101.
Gotz J. (2001) Tau and transgenic animal models. Brain Res. Brain Res.
Rev. 35, 266–286.
Gotz J., Deters N., Doldissen A., Bokhari L., Ke Y., Wiesner A.,
Schonrock N. and Ittner L. M. (2007) A decade of tau transgenic
animal models and beyond. Brain Pathol. 17, 91–103.
Grundke-Iqbal I., Iqbal K., Tung Y. C., Quinlan M., Wisniewski H. M.
and Binder L. I. (1986a) Abnormal phosphorylation of the
microtubule-associated protein tau (tau) in Alzheimer cytoskeletal
pathology. Proc. Natl Acad. Sci. USA 83, 4913–4917.
Grundke-Iqbal I., Iqbal K., Quinlan M., Tung Y. C., Zaidi M. S. and
Wisniewski H. M. (1986b) Microtubule-associated protein tau. A
component of Alzheimer paired helical ﬁlaments. J. Biol. Chem.
Gustke N., Trinczek B., Biernat J., Mandelkow E. M. and Mandelkow E.
(1994) Domains of tau protein and interactions with microtubules.
Biochemistry 33, 9511–9522.
Himmler A., Drechsel D., Kirschner M. W. and Martin D. W. Jr (1989)
Tau consists of a set of proteins with repeated C-terminal micro-
tubule-binding domains and variable N-terminal domains. Mol.
Cell. Biol. 9, 1381–1388.
Hirokawa N., Shiomura Y. and Okabe S. (1988) Tau proteins: the
molecular structure and mode of binding on microtubules. J. Cell
Biol. 107, 1449–1459.
Hong M., Zhukareva V., Vogelsberg-Ragaglia V. et al. (1998) Mutation-
speciﬁc functional impairments in distinct tau isoforms of heredi-
tary FTDP-17. Science
Hutton M., Lendon C. L., Rizzu P. et al. (1998) Association of missense
and 5¢-splice-site mutations in tau with the inherited dementia
FTDP-17. Nature 393, 702–705.
Iqbal K. and Grundke-Iqbal I. (2006) Discoveries of tau, abnormally
hyperphosphorylated tau and others of neuroﬁbrillary degenera-
tion: a personal historical perspective. J. Alzheimers Dis. 9, 219–
Iqbal K., Zaidi T., Bancher C. and Grundke-Iqbal I. (1994) Alzheimer
paired helical ﬁlaments. Restoration of the biological activity by
dephosphorylation. FEBS Lett. 349, 104–108.
Janke C., Beck M., Stahl T., Holzer M., Brauer K., Bigl V. and Arendt T.
(1999) Phylogenetic diversity of the expression of the microtubule-
associated protein tau: implications for neurodegenerative dis-
orders. Brain Res. Mol. Brain Res. 68, 119–128.
Knowles R., LeClerc N. and Kosik K. S. (1994) Organization of actin
and microtubules during process formation in tau-expressing Sf9
cells. Cell Motil. Cytoskeleton 28, 256–264.
Kosik K. S., Orecchio L. D., Bakalis S. and Neve R. L. (1989) Devel-
opmentally regulated expression of speciﬁc tau sequences. Neuron
Kren V., Pravenec M., Lu S. et al. (1997) Genetic isolation of a region of
chromosome 8 that exerts major effects on blood pressure and
cardiac mass in the spontaneously hypertensive rat. J. Clin. Invest.
Laemmli U. K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lee V. M., Balin B. J., Otvos L. Jr and Trojanowski J. Q. (1991) A68: a
major subunit of paired helical ﬁlaments and derivatized forms of
normal Tau. Science 251, 675–678.
Mandelkow E., von Bergen M., Biernat J. and Mandelkow E. M. (2007)
Structural principles of tau and the paired helical ﬁlaments of
Alzheimer’s disease. Brain Pathol. 17, 83–90.
Mawal-Dewan M., Henley J., Van de Voorde A., Trojanowski J. Q. and
Lee V. M. (1994) The phosphorylation state of tau in the devel-
oping rat brain is regulated by phosphoprotein phosphatases.
J. Biol. Chem. 269, 30981–30987.
Migheli A., Butler M., Brown K. and Shelanski M. L. (1988) Light and
electron microscope localization of the microtubule-associated tau
protein in rat brain. J. Neurosci. 8, 1846–1851.
Novak M. (1994) Truncated tau protein as a new marker for Alzheimer’s
disease. Acta Virol. 38, 173–189.
Novak M., Jakes R., Edwards P. C., Milstein C. and Wischik C. M.
(1991) Difference between the tau protein of Alzheimer paired
helical ﬁlament core and normal tau revealed by epitope analysis of
monoclonal antibodies 423 and 7.51. Proc. Natl Acad. Sci. USA
Novak M., Kabat J. and Wischik C. M. (1993) Molecular characteriza-
tion of the minimal protease resistant tau unit of the Alzheimer’s
disease paired helical ﬁlament. EMBO J. 12, 365–370.
Panda D., Samuel J. C., Massie M., Feinstein S. C. and Wilson L. (2003)
Differential regulation of microtubule dynamics by three- and four-
repeat tau: implications for the onset of neurodegenerative disease.
Proc. Natl Acad. Sci. USA 100, 9548–9553.
Schwab C., Bondada V., Sparks D. L., Cahan L. D. and Geddes J. W.
(1994) Postmortem changes in the levels and localization of
microtubule-associated proteins (tau, MAP2 and MAP1B) in the
rat and human hippocampus. Hippocampus 4, 210–225.
Sennvik K., Boekhoorn K., Lasrado R.
et al. (2007) Tau-4R sup-
presses proliferation and promotes neuronal differentiation in the
hippocampus of tau knockin/knockout mice. FASEB J. 21,
Sergeant N., Delacourte A. and Buee L. (2005) Tau protein as a dif-
ferential biomarker of tauopathies. Biochim. Biophys. Acta 1739,
2009 The Authors
Rat tau proteome |
Shea T. B., Beermann M. L., Nixon R. A. and Fischer I. (1992)
Microtubule-associated protein tau is required for axonal neurite
elaboration by neuroblastoma cells. J. Neurosci. Res. 32, 363–374.
Straiko M. M., Coolen L. M., Zemlan F. P. and Gudelsky G. A. (2007) The
effect of amphetamine analogs on cleaved microtubule-associated
protein-tau formation in the rat brain. Neuroscience 144, 223–231.
Takuma H., Arawaka S. and Mori H. (2003) Isoforms changes of tau
protein during development in various species. Brain Res. Dev.
Brain Res. 142, 121–127.
Tatebayashi Y., Iqbal K. and Grundke-Iqbal I. (1999) Dynamic regula-
tion of expression and phosphorylation of tau by ﬁbroblast growth
factor-2 in neural progenitor cells from adult rat hippocampus.
J. Neurosci. 19, 5245–5254.
Wischik C. M., Novak M., Edwards P. C., Klug A., Tichelaar W. and
Crowther R. A. (1988a) Structural characterization of the core of
the paired helical ﬁlament of Alzheimer disease. Proc. Natl Acad.
Sci. USA 85, 4884–4888.
Wischik C. M., Novak M., Thogersen H. C., Edwards P. C., Runswick
M. J., Jakes R., Walker J. E., Milstein C., Roth M. and Klug A.
(1988b) Isolation of a fragment of tau derived from the core of the
paired helical ﬁlament of Alzheimer disease. Proc. Natl Acad. Sci.
USA 85, 4506–4510.
Zilka N., Filipcik P., Koson P., Fialova L., Skrabana R., Zilkova M.,
Rolkova G., Kontsekova E. and Novak M. (2006) Truncated tau
from sporadic Alzheimer’s disease sufﬁces to drive neuroﬁbrillary
degeneration in vivo. FEBS Lett. 580, 3582–3588.
Zilka N., Kontsekova E. and Novak M. (2008) Chaperone-like anti-
bodies targeting misfolded tau protein: new vistas in the immu-
notherapy of neurodegenerative foldopathies. J. Alzheimers Dis.
2009 The Authors
1176 | J. Hanes