(Received for publication, March 2, 1995; and in revised form, May 12, 1995)
From the
Paired helical filaments (PHFs) are the major structural
elements of Alzheimer's disease neurofibrillary lesions, and
these filaments are formed from hyperphosphorylated brain tau known as
PHF-tau. Recent studies showed that many previously identified
phosphorylated residues in PHF-tau also are phosphate acceptor sites in
fetal and rapidly processed adult brain tau. However, Ser has been suggested to be uniquely phosphorylated in PHF-tau and a
key regulator of the binding of tau to microtubules. For these reasons,
we generated a monoclonal antibody (12E8) specific for phosphorylated
Ser
and showed that 12E8 binds to PHF-tau, rat and human
fetal brain tau, as well as to rapidly processed adult rat and
biopsy-derived human brain tau. Further, phosphorylation at Ser
was developmentally regulated, and endogenous brain phosphatases
rapidly dephosphorylated Ser
in biopsy-derived brain tau
isolates. Finally, the phosphorylation of Ser
did not
eliminate the binding of tau to microtubules. Thus, we speculate that
the binding of tau to microtubules is regulated by phosphorylation at
multiple sites and that the generation of PHF-tau in Alzheimer's
disease results from the reduced efficiency of phosphatases leading to
the incremental accumulation of hyperphosphorylated tau.
Tau protein is the major component of paired helical filaments
(PHFs) ()in Alzheimer's disease (AD) neurofibrillary
tangles and related neurofibrillary
lesions(1, 2, 3, 4, 5, 6) .
It is expressed predominantly in axons where its major function is to
bind to and stabilize microtubules
(MTs)(7, 8, 9) . The MT binding domains of
tau are 31- or 32-amino acid motifs repeated three or four times in the
carboxyl-terminal half of the molecule. The presence or absence of 29-
or 58-residue amino-terminal inserts in conjunction with three or four
MT binding repeats gives rise to six tau isoforms in adult human brain (10, 11) , but only the shortest (``fetal'')
isoform (three MT binding repeats, no amino-terminal inserts) is
present in developing human brain(11) .
PHFs are comprised
of all six tau isoforms in a hyperphosphorylated state (PHF-tau)
relative to normal tau in adult brain(12) , and PHF-tau has a
greatly reduced ability to bind to MTs(13, 14) . This
is due to hyperphosphorylation, since dephosphorylated PHF-tau binds
MTs like normal tau. Thus, hyperphosphorylation may be a key event in
the transformation of normal brain tau into
PHF-tau(5, 6) , and PHFs may result from the
self-association of hyperphosphorylated tau through its MT-binding
repeats(15, 16) . Mass spectrometry and immunological
studies have identified a number of phosphorylation sites in PHF-tau,
some of which are not phosphorylated in tau isolated from postmortem
adult human
brains(13, 17, 18, 19, 20, 21) .
Many of these sites are Ser/Thr-Pro motifs and, except for
Ser, are located outside the MT-binding region.
Ser
is located in the first MT-binding repeat of
tau(18) . Except for Ser
, many of the
phosphorylation sites in PHF-tau also are phosphorylated in a
significant fraction of fetal brain
tau(13, 17, 18, 19) . Further,
recent studies on adult rat brain tau (21, 22) and
adult human brain tau isolated from biopsy samples (23) have
demonstrated that some phosphorylation sites previously identified in
PHF-tau and fetal tau also are phosphorylated in a fraction of adult
tau. However, Ser
has been reported to be phosphorylated
only in PHF-tau(18, 19) .
Tau protein can be
phosphorylated in vitro at many of the above sites by
proline-directed protein kinases such as mitogen-activated protein
kinase(24, 25, 26) , glycogen synthase kinase
3 (27, 28, 29) , and cyclin-dependent kinase
5(30, 31, 32) . In addition, cyclic
AMP-dependent protein kinase(33) ,
Ca/calmodulin-dependent protein kinase II (34) , and protein kinase C (35) phosphorylate tau at
specific sites in vitro, but among these sites, only
Ser
and Ser
are phosphorylated in PHF-tau.
Although none of the above protein kinases has been reported to
phosphorylate Ser
, Ser
has been reported to
be phosphorylated by a novel 35/41-kDa kinase partially purified from
brain(36) .
Since the latter study emphasized the critical
importance of Ser in regulating the binding of tau to MTs
and the pivotal role that Ser
may play in the
pathogenesis of PHF-tau in AD(36) , we explored this issue
further using a novel monoclonal antibody (MAb) raised to a synthetic
phosphopeptide spanning amino acid residues 257-270 in human tau
with a phosphoserine at position 262. Using this novel MAb, we show
here that Ser
is phosphorylated in PHF-tau as well as in
normal brain tau and that phosphorylation at Ser
alone
cannot account for the inability of PHF-tau to bind to MTs.
Figure 1:
RIA studies of the specificity of 12E8.
The phosphorylated (filledcircles) or
non-phosphorylated (opencircles) synthetic peptides
spanning the tau sequence including Ser were compared by
competitive RIA. The
I-labeled ligand was prepared using
chloramine-T and is present in the reaction mixtures at approximately 4
nM. Determinations were made in triplicate, and they varied
less than 10% from each other.
Figure 2:
Epitope mapping of 12E8. Wild type (WT) recombinant tau expressed from cDNA clone htau24 and
mutated at Ser (Ala
/Ser
),
Ser
(Ser
/Ala
) or Ser
and Ser
(Ala
/Ala
) were
immunoblotted with antiserum 134 (panelA) or MAb
12E8 (panelB) following incubation without
(-) or with (+) a rat brain extract, resulting in the
phosphorylation of tau. Antiserum 134 is not
phosphorylation-dependent.
Figure 3:
Studies of PHF-tau and normal fetal and
adult human tau with 12E8. Western blots comparing the site and extent
of phosphorylation in the different tau preparations are shown in panelsA-D. Biopsy-derived human tau (B),
autopsy-derived fetal tau (F
), autopsy-derived adult human tau
(A
), and PHF-tau (PHF
) were loaded onto 10% SDS-PAGE gels for
immunoblot analysis. B
-B
denote
tau from three different biopsy samples. The antibodies used here are
shown on each blot. To obtain comparable levels of T14/T46
immunoreactive tau (i.e. as shown in E) in different
tau preparations, the following amounts of total protein from each
preparation were loaded: F
, 2.5 µg; A
, 5 µg;
PHF
, 2 µg; and B
, 10 µg. The same amount of PHF
was loaded for all antibodies. However, twice the amount of F
and
A
and three times the amount of B
were loaded for PHF1, 12E8,
and AT8. PanelsE-H show quantitative data from
PhosphorImager analysis of the immunoblots shown in panelsA-D using
I-labeled goat anti-mouse
IgG. The extent of phosphorylation of F
, A
, and B
relative to PHF
was calculated by taking into account the amount
of protein loaded in each lane. The extent of phosphorylation
in F
, A
, and B
for each lane was normalized to
PHF
(for the antibodies PHF1, 12E8, and
AT8).
Figure 4:
Phosphorylation of Ser in
PHF-tau as well as in fetal and adult human tau. Fetal and
biopsy-derived normal human tau and PHF-tau were digested with NTCB,
and the digests were separated on 12% SDS-PAGE gels followed by
transfer to nitrocellulose. The MAbs used to probe the tau digests are
shown. Tau indicates the presence of undigested tau, N marks the position of the amino-terminal fragments, whereas C labels the carboxyl-terminal fragments. 12E8 only binds to the
amino-terminal fragments of tau.
Figure 5:
Western blot analysis of BI-tau
(supernatant) and BC-tau (pellet). BI-tau (in lanes marked S) and BC-tau (in lanes marked P) were
subjected to immunoblot analysis (panelsA-D).
SP
and S
P
denote BI and
BC-tau from two different biopsies. The amount of protein loaded for
detection by T14/46 (panelA) in the supernatant (S) fraction was three times that for the pellet (P)
fraction. To obtain comparable levels of T14/46 immunoreactive tau (as
shown in E), we loaded 2 µg of PHF
(for all
antibodies) and 5 µg of A
. Approximately 10 µg of B
from the pellet fraction were loaded per lane in panelA. The amount of A
and B
loaded for the
antibodies PHF1, 12E8, and AT8 was two times as that for T14/46. PanelsE-H show the quantitative data using a
PhosphorImager with
I-labeled goat anti-mouse IgG as
secondary antibody.
Figure 6:
Western blot analysis of fetal, postnatal,
and adult rat tau. Nitrocellulose replicas were prepared from 7.5%
SDS-PAGE gels, and representative Western blots are shown in panelsA and B. To obtain comparable signals of T46/49
immunoreactive tau, we loaded in lane1, embryonic
day 18 (E18, 15 µg); lane2, post-natal day 7
(P7, 15 µg); lane3, postnatal day 11 (P11, 25
µg); lane4, postnatal day 19 (P19, 25 µg); lane5, postnatal day 25 (P25, 40 µg); lane6, adult 3-month-old (3 M, 40 µg); and lane7, adult 20-month-old (20 M, 40
µg). The antibodies used here are labeled above each blot. The amount of protein loaded for the antibody 12E8 was
three times that mentioned above for T46/49. PanelsC and D show quantitative data from PhosphorImager analysis
of the immunoblots shown in panelsA and B.
The decrease in phosphorylation was normalized to 100% for E18.
Standard deviations were generated from quadruplicate samples, and the
quantitation was generated using I-labeled goat
anti-mouse IgG.
Since many phosphorylation sites in adult tau are
rapidly dephosphorylated by endogenous brain phosphatases during
postmortem or postsurgical delays(23) , we simulated such
conditions by allowing freshly excised human brain biopsy samples to
remain at room temperature for varying lengths of time as
described(23) . The tau proteins isolated from these samples
were then analyzed by immunoblotting using many of the anti-tau
antibodies described above (Fig.7). Fig. 7A shows the profile of tau proteins in these samples labeled with
the T14/46 mixture of MAbs. Consistent with previous
studies(23) , Fig.7, C and G, shows
that biopsy-derived adult human brain tau was rapidly dephosphorylated
at Ser at room temperature. In fact, after only 1 h of
simulated postsurgical delay, the extent of phosphorylation of tau at
Ser
was reduced by over 90% (Fig.7G).
Notably, Ser
was much more rapidly dephosphorylated than
Ser
/Ser
or
Ser
/Thr
. After only a 5-min postsurgical
delay, the intensity of the immunobands detected by 12E8 was reduced to
a far greater extent (i.e. by about 70%) than that produced by
other phosphate-dependent MAbs (i.e. compare Fig.7B, 7D, 7F, and 7H with
7C and 7G).
Figure 7:
Dephosphorylation of human tau in
situ. Western blot analysis of the time course of
dephosphorylation of biopsy-derived brain tau during defined
post-surgical intervals of 0-60 min as indicated below the lanes in panelsA-D. Biopsy
brain samples were left at room temperature for periods of time
extending from 5 to 60 min. The antibodies used for immunoblotting are
identified above each panel. All the lanes were loaded with F, A
, and PHF
. PanelsE-H show quantitative data from PhosphorImager
analysis of the immunoblots shown in panelsA-D. The decrease in phosphorylation at each site
was normalized to time zero (i.e. no post-surgical
delay).
The phosphorylation of biopsy-derived
tau and PHF-tau at Ser was also demonstrated by
immunohistochemistry (Fig.8). Fig. 8A shows
intense 12E8 immunoreactivity in processes throughout the cortical
neuropil of a biopsy sample while neuronal perikarya stand out as 12E8
negative oval or pyramidal profiles. In sections from postmortem AD
neocortex (Fig.8B), 12E8 intensely labeled the PHF-tau
accumulations in perikaryal neurofibrillary tangles, neuropil threads,
and dystrophic neurites, much like previously described
phosphorylation-dependent anti-tau antibodies (e.g. see
results obtained with PHF1 in Fig.8C).
Figure 8:
Immunohistochemical localization of
tau-phosphorylated Ser with 12E8. Immunohistochemical
staining of tissue sections obtained from biopsy control (A)
and autopsy AD human brain samples. In panelsA and B, sections had been incubated with 12E8, and in panelC, sections had been incubated with PHF1. All brain
sections were obtained from temporal lobe. The magnification in panelsA-C is the same, and the bar in panelC = 50
µm.
We have used a novel MAb (12E8) to assess the role of
phosphorylated Ser in the pathogenesis of PHF-tau. The
specificity of 12E8 for tau phosphorylated at Ser
was
demonstrated by RIA and Western blot analyses. Since the sequences
surrounding Ser
and Ser
are similar, we
performed additional studies using recombinant tau proteins harboring
Ser to Ala mutations at positions 262 and/or 356, which showed that
12E8 recognizes recombinant tau when phosphorylated at
Ser
, Ser
, or at both positions. Since 12E8
recognized PHF-tau, fetal brain tau, and biopsy-derived adult brain
tau, we probed NTCB cleavage products of tau and demonstrate that only
Ser
is phosphorylated in tau from normal and AD brain.
The recognition of PHF-tau on immunoblots and the intense staining
of neurofibrillary lesions in AD brain by immunohistochemistry with
12E8 confirm that Ser is phosphorylated in
PHF-tau(18) ; however, contrary to previous
findings(18) , we also found that Ser
is a site
of phosphorylation in normal fetal and adult human and rodent tau. The
demonstration here that Ser
is rapidly dephosphorylated
by brain phosphatases appears to account for the discrepancy between
the present and previous
studies(18, 19, 20) . Our results indicate
that of the phosphorylation sites in brain tau studied to date,
Ser
is the most labile since 70% of the phosphate was
lost within 5 min of postsurgical delay, compared with 10% for
Ser
/Ser
and 50% for
Ser
/Thr
. Further, we also show that the
phosphorylation of Ser
in normal brain tau is
developmentally regulated and that only a fraction of biopsy-derived
tau is phosphorylated at Ser
compared with PHF-tau.
Similar results also have been obtained with anti-tau antibodies AT8,
AT180, AT270, T3P, and PHF1, which recognize specific Ser/Thr-Pro sites
in tau when
phosphorylated(13, 17, 21, 22, 23) .
Since 12E8 is the first antibody to a phosphorylation site in tau that
is not of the Ser/Thr-Pro type, the present data indicate that
proline-directed kinases do not fully account for the normal or the
abnormal phosphorylation of tau. Although a novel 35/41-kDa protein
kinase partially purified from brain phosphorylates tau at Ser
in vitro, both cyclic AMP-dependent protein kinase and
Ca
/calmodulin-dependent protein kinase II
phosphorylate recombinant tau at this site in vitro. (
)Thus, multiple protein kinases may phosphorylate tau at
this site in normal and AD brains. Further, reduced phosphatase
activity may play an important role in PHF-tau
formation(22, 23) . Although the protein phosphatases
that act on the Ser
site are unknown, preliminary results
indicate that protein phosphatase 2A dephosphorylate tau at
Ser
. (
)
The findings described here serve to
further clarify the pathogenesis of PHF-tau by demonstrating that
phosphorylation of Ser is not sufficient to eliminate the
binding of tau to MTs. Indeed, they suggest that the simultaneous
phosphorylation of multiple sites may be required for the disruption of
the interactions between tau and MTs. Thus, the challenge for the
future will be to identify the phosphorylation sites that are most
critical for regulating the binding of tau to MTs, as well as the
protein kinases and protein phosphatases that modulate the state of
phosphorylation of tau in both normal and AD brain.