(Received for publication, September 15, 1994; and in revised form, December 6, 1994)
From the
Microtubule-associated protein is abnormally
hyperphosphorylated in the brain of patients with Alzheimer disease and
in this form is the major protein subunit of the paired helical
filaments (PHF), the most prominent lesion of the disease. In this
study the dephosphorylation of sparingly soluble PHF, PHF II-
by
brain protein phosphatase (PP)-2A
and PP-2B, and the
resulting biochemical, biological, and structural alterations were
investigated. Both of the phosphatases dephosphorylated PHF II-
at
the sites of Ser-199/Ser-202 and partially dephosphorylated it at
Ser-396/Ser-404; in addition, PHF II-
was dephosphorylated at
Ser-46 by PP-2A
and Ser-235 by PP-2B. The relative
electrophoretic mobility of PHF II-
increased after
dephosphorylation by either enzyme. Divalent cations, manganese, and
magnesium increased the activities of PP-2A
and PP-2B
toward PHF II-
. Dephosphorylation both by PP-2B and PP-2A
decreased the resistance of PHF II-
to proteolysis by the
brain calcium-activated neutral proteases (CANP). The ability of PHF
II-
to promote the in vitro microtubule assembly was
restored after dephosphorylation by PP-2A
and PP-2B.
Microtubules assembled by the dephosphorylated PHF II-
were
structurally identical to those assembled by bovine
used as a
control. The dephosphorylation both by PP-2A
and PP-2B
caused dissociation of the tangles and the PHF; some of the PHF
dissociated into straight protofilaments/subfilaments. Approximately
25% of the total
was released from PHF on dephosphorylation by
PP-2A
. These observations demonstrate that PHF II-
is
accessible to dephosphorylation by PP-2A
and PP-2B, and
dephosphorylation makes PHF dissociate, accessible to proteolysis by
CANP, and biologically active in promoting the assembly of tubulin into
microtubules.
Microtubules are required for the integrity of the neuronal
cytoskeleton and the axonal transport. Alzheimer disease (AD) ()is characterized by replacement and displacement of
microtubules by paired helical filaments (PHF) in certain selected
brain neurons, especially in hippocampus. PHF exist as neurofibrillary
tangles in neuronal cell bodies, as neuropil threads in the dystrophic
neurites of affected neurons, and in degenerating neurites surrounding
the extracellular amyloid in neuritic (senile) plaques.
Microtubule-associated protein
is the major protein subunit of
PHF (Grundke-Iqbal et al., 1986a; Iqbal et al., 1989;
Lee et al., 1991).
in AD brain, especially PHF, is
abnormally phosphorylated (Grundke-Iqbal et al., 1986b; Iqbal et al., 1986, 1989; Lee et al., 1991). The abnormally
phosphorylated
from AD brain contains 5-9 mol of
phosphate/mol of the protein, which is about three to four times the
level of phosphate in normal brain
(Köpke et al., 1993). PHF-
is phosphorylated at multiple sites
(Iqbal et al., 1989). So far 21 abnormal phosphorylation sites
of PHF-
(all Ser/Thr sites) have been identified either by
phosphorylation-dependent antibodies (Grundke-Iqbal et al.,
1986b; Iqbal et al., 1989; Brion et al., 1991; Lee et al., 1991; Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Kanemaru et al., 1992) or by mass
spectrometry (Hasegawa et al., 1992; Morishima-Kawashima et al., 1995). Most of the phosphates are clustered at two
sites, one amino-terminal (Ser-198 to Thr-217) and one
carboxyl-terminal (Ser-396 to Ser-422) to the microtubule-binding
repeat domains (Gln-244 to Gly-367); numbering according to the largest
isoform,
(Goedert et al., 1989). As a
result of the abnormal hyperphosphorylation, the apparent molecular
weight of PHF-
on SDS-polyacrylamide gels is higher than that of
normal
(Grundke-Iqbal et al., 1986b; Iqbal et
al., 1989; Lee et al., 1991). Furthermore, PHF-
does
not promote the assembly of tubulin into microtubules unless it is
dephosphorylated prior to its interaction with tubulin (Iqbal et
al., 1994).
from AD brain can be biochemically isolated
into three populations (Köpke et al.,
1993): (i) non-abnormally phosphorylated cytosolic
(C-
),
(ii) soluble abnormally phosphorylated
(AD P-
) and, (iii)
abnormally phosphorylated
polymerized into PHF (PHF-
). Based
on the solubility, PHF-
can be further classified into two
species: PHF I-
and PHF II-
. PHF I-
is readily soluble
in 2.0% SDS, whereas PHF II-
requires ultrasonication and heating
in SDS for extractions (Iqbal et al., 1984). Pathologically,
these species of
may reflect different stages of neuronal
degeneration in AD brain, that is, normal
(C-
) first becomes
abnormally hyperphosphorylated (AD P-
) and then by a presently
unknown mechanism becomes polymerized into PHF (PHF-
); unlike AD
P-
and PHF I-
, PHF II-
also becomes partly ubiquitinated
(Grundke-Iqbal et al., 1988; Morishima-Kawashima et
al., 1993).
Phosphoseryl and phosphothreonyl protein
phosphatases, which are classified into four major types, i.e. PP-1, PP-2A, PP-2B, and PP-2C (for review, see Cohen(1989)), are
present in significant amounts in human brain (Gong et al.,
1993). We have previously found that PP-2C is not active toward any
abnormal sites of AD P-, whereas PP-1 is only effective toward two
of the epitopes investigated (Gong et al., 1994b). Therefore,
PP-2A and PP-2B are the most probable candidate phosphatases involved
in the abnormal phosphorylation of
in AD and for this reason were
chosen for this study.
Previous studies have shown that AD P-
can be dephosphorylated by PP-2A
(equally well by
PP-2A
and PP-2A
) and PP-2B at several abnormal
phosphorylation sites (Gong et al., 1994a and 1994c), implying
that the neurodegenerative changes in AD in their early stage might be
reversible.
In the present study, dephosphorylation of PHF II-,
a late stage of Alzheimer neurofibrillary pathology (Bancher et
al., 1989; Köpke et al., 1993) by
brain PP-2A
and PP-2B and the resulting biochemical,
biological, and structural alterations were investigated. PHF II-
were found to be partially accessible to dephosphorylation by
PP-2A
and PP-2B, and treatment with these phosphatases
caused dissociation of PHF/tangles, decreased the protease resistance,
and restored the microtubule assembly-promoting activity of PHF
II-
.
Human brains employed for this study were obtained within 6 h
postmortem and stored frozen at -75 °C until used. Protein
phosphatase 2A and 2B were purified from bovine brain
according to the methods described by Cohen et al.(1988) and
Sharma et al.(1983), respectively. CANP (a mixture of micro-
and millimolar calcium-dependent enzymes) was purified from calf brain
by the procedure described previously (Malik et al., 1983).
Polyclonal antibody 92e and 102c were raised as reported previously
(Grundke-Iqbal et al., 1988; Iqbal et al., 1989).
Monoclonal antibodies
-1 and PHF-1 were provided by Drs. L. I.
Binder (Binder et al., 1985) and S. Greenberg (Greenberg et al., 1992), respectively. SMI31, SMI33, and SMI34 were
purchased from Sternberger Monoclonals Inc., Baltimore, MD. Alkaline
phosphatase-conjugated anti-mouse and anti-rabbit IgG were purchased
from Sigma.
I-Labeled donkey anti-rabbit whole antibody
was from Amersham (Arlington, IL).
Normal human was purified as described previously
(Köpke et al., 1993) from 35-45%
ammonium sulfate precipitates of the 200,000
g brain
supernatant, followed by acid treatment (pH 2.7) and chromatography on
a phosphocellulose column (Cellulose phosphate P11, Whatman).
Figure 2:
Dephosphorylation of PHF II- and AD
P-
by various concentrations of PP-2A
and PP-2B.
Western blots of PHF II-
(a, 2.0 µg/lane) and AD
P-
(b, 0.5 µg/lane) were carried out using
-1 as
primary antibody after incubation either without (a and b,
lane 1) or with PP-2B (a, lanes 2-4; b, lanes
2-5) or with PP-2A
(a, lanes 5-7; b, lanes 6-9) at 37 °C for 45 min. In a,
the concentrations of PP-2B for lanes 2-4 were 1.0
units/ml, 2.5 units/ml, and 5.0 units/ml, respectively, whereas those
of PP-2A
for lanes 5-7 were 0.5 unit/ml, 1.0
unit/ml, and 2.0 units/ml, respectively. The molecular mass (kDa)
standards are shown at the left of a. In b,
PP-2B for lanes 2-5 was used at concentrations of 0.36
unit/ml, 0.91 unit/ml, 1.82 units/ml, and 3.63 units/ml, whereas
PP-2A
for lanes 6-9 was 0.1 unit/ml, 0.25
unit/ml, 0.5 unit/ml, and 1.0 unit/ml, respectively. A higher
concentration of the phosphatases was required to dephosphorylate PHF
II-
than AD P-
.
Figure 1:
Dephosphorylation of PHF II- by
PP-2A
and PP-2B. Western blots of PHF II-
with
different antibodies were carried out after incubation either without (lane 1) or with 5.0 units/ml PP-2B (lane 2) or with
2.0 units/ml PP-2A
(lane 3) at 37 °C for 45
min as described under ``Materials and Methods.'' Lane 4 is untreated normal human
as a control. Six
phosphorylation-dependent, site-specific monoclonal antibodies and one
polyclonal anti-
antibody were employed for immunoblotting as
shown beneath each panel. The epitope of each antibody is described in Table 1. Molecular mass (kDa) standards are indicated at the left of a. The amounts of protein/lane were 2 µg
for 92e and
-1, 3 µg for PHF-1 and 102c, and 5 µg for
SMI31, SMI33, and SMI34, respectively. Both PP-2A
and PP-2B
dephosphorylated Ser-199/Ser-202 (
-1) and partially
dephosphorylated Ser-396/Ser-404 (SMI31 and PHF-1). PP-2A
and PP-2B also dephosphorylated Ser-46 (102c) and Ser-235
(SMI31), respectively. In addition, dephosphorylation by either
phosphatases changed the conformation (SMI34) and shifted the mobility
(92e) of
in PHF II. The prominent immunostaining of the PHF high
molecular mass smear by antibody 102e (Ser 46) in b, lane 3,
suggests that the smear selectively contains
isoforms with the
amino-terminal insert(s). Polypeptides with a mass of less than 43 kDa
represent the degraded
.
Figure 3:
Effects of divalent cations and other
effectors on the dephosphorylation of PHF II- by PP-2A
(a) and PP-2B (b). Western blots of PHF
II-
(2 µg/lane) with antibody
-1 were carried out after
incubation of the substrate either without phosphatase (a and b, lane 1) or with phosphatase (PP-2A
, 2 units/ml;
PP-2B, 5 units/ml) and different effectors (a and b, lanes
2-11). The molecular mass (kDa) markers are shown at the left of the panels. In a and b, the reaction
mixture for lanes 2-4 contained 0.01 mM, 0.1
mM, or 2.0 mM MnCl
and for lanes
5-7 contained 0.1 mM, 1.0 mM, or 10.0
mM MgCl
. Lanes 8-10 in a were with 1.0, 10.0, and 100.0 µM polylysine, whereas lanes 8-10 in b were with 1.0 mM CaCl
, 1.0 µM calmodulin; 1.0 mM CaCl
, 1.0 µM calmodulin, 1.0 mM MnCl
; and 1.0 mM CaCl
, 1.0
µM calmodulin, 1.0 mM MgCl
,
respectively. Lane 11 was with 5.0 mM EDTA in a and 5.0 mM EGTA in b, respectively.
Mn
and Mg
activated both
phosphatases, whereas Ca
/calmodulin stimulated PP-2B.
Polylysine minimally promoted the PP-2A
activity.
Figure 4:
Dephosphorylation-induced CANP proteolysis
of PHF II-. PHF II-
(2 µg/lane) was incubated with 0.25
unit/ml CANP for different times (as indicated beneath each lane)
untreated (a) or dephosphorylated by PP-2A
(b) or by PP-2B (c) as described under
``Materials and Methods.'' After incubation with CANP,
samples were subjected to Western blotting with
polyclonal
antibody 92e. Dephosphorylation of PHF II-
by either enzymes
decreased its resistance to CANP proteolysis. The molecular mass (kDa)
markers are shown at the left of the
panels.
Figure 5:
Effect of dephosphorylation of PHF
II- by PP-2A
and PP-2B on its microtubule
assembly-promoting activity. PHF II-
was dephosphorylated and then
extracted as described under ``Materials and Methods.''
Microtubule assembly was carried out by incubating at 37 °C rat
brain tubulin (3.0 mg/ml) with bovine
or PHF II-
(0.2
mg/ml). Microtubule assembly-promoting activity was increased after
phosphatases treatment. Curves show microtubule assembly in the
presence of normal
, PHF dephosphorylated with PP-2A
and PP-2B, PHF untreated with any phosphatase, and assembly with
tubulin alone.
Figure 6:
Electron micrographs showing the products
of microtubule assembly negatively stained with phosphotungstic acid.
Aliquots of each sample (from Fig. 5) were taken at steady state
of polymerization and stained negatively with 2% phosphotungstic acid
as described under ``Materials and Methods.'' Large numbers
of microtubules from tubulin were observed in the presence of bovine
and PP-2A
- and PP-2B-treated PHF (a, c, and d, respectively); no microtubules were seen in the presence of
nondephosphorylated PHF II-
(b), and an occasional
microtubule could be observed in the tubulin alone control (not shown).
No ultrastructural differences were observed among microtubules
assembled with the bovine control
and the phosphatases-treated
PHF II-
. Bar, 0.5 µm.
Figure 7:
Electron micrographs of PHF II-
before and after phosphatases treatment. Incubation of PHF II-
was
carried out at 37 °C for 45 min (a, b (two
panels), and c (three panels)) or 3 h (d, e, and f) either in the absence (a and d) or the
presence of PP-2A
(b and e) or PP-2B (c and f). The dephosphorylation was terminated by
the addition of 100 mM of phosphate buffer (pH 7.5), and the
grids were prepared as described under ``Materials and
Methods.'' Marked structural changes could readily be seen after
3-h dephosphorylation by either enzyme. Bars, 0.1
µm.
One of the most characteristic brain lesions of AD is the
formation of PHF in the affected neurons. The abnormal phosphorylation
of probably precedes its polymerization into PHF (Bancher et
al., 1989; Köpke et al., 1993).
Previously, we have demonstrated that this pre-PHF abnormal
, AD
P-
, can be dephosphorylated at several abnormal phosphorylation
sites by PP-2A
and PP-2B (Gong et al., 1994c,
1994a) and that both of these enzymes are localized in neurons,
including neurons predilected for neurofibrillary tangles (Pei et
al., 1994). Furthermore, Drewes et al.(1993) have
reported that the
extracted from PHF by detergents and made
soluble can be dephosphorylated at the AT8 site (this antibody
recognizes Ser-199/Ser-202 when phosphorylated) by PP-2A and PP-2B. In
the present study, we have investigated whether
polymerized into
PHF/neurofibrillary tangles is also accessible to protein phosphatases
and have examined the effect of dephosphorylation on the structure,
proteolysis, and the biological activity of
in PHF. We found (i)
that PHF II-
can be dephosphorylated at Ser-46 by
PP-2A
, at Ser-235 by PP-2B, and at Ser-199/Ser-202 and
partially at Ser-396/Ser-404 by both PP-2A
and PP-2B; (ii)
that the dephosphorylation of PHF II-
by either phosphatase
decreases its resistance to proteolysis by CANP; (iii) that the
dephosphorylation of PHF II-
with either phosphatase restores its
biological activity as determined by the microtubule assembly-promoting
activity; and (iv) that the dephosphorylation leads to a dissociation
of the neurofibrillary tangles and individual PHF into straight
protofilaments and release of
. These data suggest that not only
soluble abnormally phosphorylated
but also the
polymerized
into PHF is accessible, although to a lesser degree, to the protein
phosphatases PP-2A
and PP-2B, and on dephosphorylation with
these enzymes the structure of PHF dissociates and
becomes
biologically active and can be proteolyzed by CANP.
Compared with AD
P-, PHF II-
is less amenable to dephosphorylation by
PP-2A
and PP-2B. The phosphates at
-1 epitope in AD
P-
were readily removed by 0.1 unit/ml PP-2A
or 0.36
unit/ml PP-2B, whereas for PHF II-
, 2.0 units/ml PP-2A
or 5.0 units/ml PP-2B were required for maximal dephosphorylation
at the same epitope. Pathologically, AD P-
and PHF II-
may
reflect, respectively, an early stage and a late stage of AD
neurofibrillary degeneration. The structural state of
in PHF
might be responsible for its decreased accessibility to the
phosphatases. The same reason may also explain why AD P-
can be
dephosphorylated at the abnormal sites Ser-396/Ser-404 by PP-2A and
PP-2B (Gong et al., 1994a, 1994c), whereas PHF II-
can be
only partially dephosphorylated at these sites. The exact reason for
the relative resistance of Ser-396/Ser-404 sites for dephosphorylation
in PHF II-
by PP-2A
, and PP-2B is at present not
known. It is likely that the carboxyl-terminal region of the abnormally
phosphorylated
is buried into the PHF polymer and is less
accessible to the phosphatases.
The effect of divalent cations on
the phosphatase activities showed that Mn stimulated
PP-2A
and PP-2B at the concentration of 0.01 mM,
whereas 1.0 mM Mg
was required for
comparable activities. These concentrations of Mn
and
Mg
are in the physiological range (Friberg et
al., 1986). The stimulation of the phosphatase activities by these
metals might have physiological and therapeutic significance.
Using
soluble AD P- as a substrate and rat brain phosphatase as an
enzyme source, we (Gong et al., 1994a, 1994c) demonstrated
previously that in vitro PP-2B was more active than PP-2A.
Similar results were reported by Drewes et al. (1993). In the
present study, we found that bovine brain PP-2A
was more
active than PP-2B toward PHF II-
, suggesting that both PP-2A
and PP-2B might play a role in the abnormal phosphorylation of
and that the differences between the previous and the present
studies might be related to the use of different substrates and
possibly different enzyme sources.
Although the mechanism for PHF
formation and accumulation as tangles remains largely unknown, one can
postulate that an impaired proteolysis of abnormally phosphorylated
might be related to this pathological processing. Previous
studies have shown that PHF is extremely protease-resistant
(Grundke-Iqbal et al., 1988; Wischik et al., 1988).
In the present study we have found that dephosphorylation of PHF
II-
by PP-2A
and PP-2B increases its proteolysis by
CANP. These findings suggest that abnormal phosphorylation of PHF-
might be involved in its increased resistance to CANP digestion, and
this abnormal phosphorylation-induced inhibition of
proteolysis
by CANP can be reversed by the dephosphorylation of the abnormal
by PP-2A
and PP-2B. We also found that dephosphorylated AD
P-
was degraded significantly more rapidly and extensively than
PHF II-
(data not shown). These differences in the CANP
proteolysis of AD P-
and PHF II-
might be related to the
nonpolymeric state and a more complete dephosphorylation of the former
than the latter.
Interestingly, it was also found that normal human
was rapidly digested by the CANP used in this study but not by
that purchased from Sigma (data not shown). Johnson et
al.(1989) also reported that
purified from total brain
heat-stable fraction was resistant to degradation by the CANP from
Sigma. The CANP from Sigma was purified from rabbit skeletal muscle and
only contained the millimolar type of CANP, whereas the CANP used in
the present study was purified from calf brain and contained both
millimolar and micromolar types of CANP. Thus, the difference in the
activities of CANP toward
between the Sigma enzyme and the enzyme
used in the present study might be either due to the tissue specificity
or the type specificity or both; tissue-specific CANP have been
reported recently (Sorimachi et al., 1994).
Previous
studies have shown that soluble abnormally phosphorylated and PHF
II-
isolated from AD brain had no microtubule assembly activity.
However, when this
was extensively dephosphorylated by alkaline
phosphatase, the microtubule assembly-promoting activity was restored
(Alonso et al., 1994; Iqbal et al., 1994). In the
present study, we found that partial dephosphorylation of PHF II-
by protein phosphatase 2A
or 2B also restored its
biological activity in promoting the in vitro microtubule
assembly. The assembly activity of the PP-2A
-treated sample
as determined by the turbidimetric changes was much higher than that of
the PP-2B-treated sample. These findings suggest (i) that the
phosphorylation of
at Ser-235, a site dephosphorylated by PP-2B,
and not by PP-2A
, might not be involved in the microtubule
assembly-promoting activity; and (ii) that one or more selective sites
that are dephosphorylated by PP-2A
but not by PP-2B might
enhance the microtubule assembly activity. The nature of these
differences, which will be investigated in a separate study, is, at
present, not known. Although the turbidity is significantly higher in
the PP-2A
-treated sample than PP-2B-treated sample, no
obvious difference in the structure of microtubules was seen by the
negative stain electron microscopy.
PHF are ultrastructurally
distinct from elements of normal neuronal cytoskeleton, i.e. microtubules and neurofilaments. PHF have a diameter of
22-24 nm, which narrows to 10 nm at every 80-nm interval
(Kidd, 1964; Wisniewski et al., 1984). In this study, we
observed that dephosphorylation of PHF by either PP-2A
or
PP-2B dissociated PHF tangles, and this dissociation became more
significant with the increase in dephosphorylation. During the first 45
min of dephosphorylation, dissociation of PHF from the neurofibrillary
tangles and a decrease in the discernability of the twists of PHF were
observed. After the dephosphorylation for 3 h, a fewer number of the
tangles and PHF and a concomitant release of
was observed.
Furthermore, an increasing number of PHF were found to lose their twist
and were dissociated into straight protofilaments. Although the role of
phosphorylation in the polymerization of
into PHF is not
understood, the present study suggests that dephosphorylation by
PP-2A
and PP-2B might reverse this lesion.
In
conclusion, the present study reveals that in PHF II is
biologically inactive and protease-resistant. However, PHF in
neurofibrillary tangles are accessible to dephosphorylation at some
sites by PP-2A
and PP-2B. Dephosphorylation of PHF-
restores the microtubule assembly-promoting activity, increases the
proteolysis by CANP, and dissociates the neurofibrillary tangles and
the PHF. Dephosphorylation might inhibit and reverse the
neurofibrillary degeneration in AD brain.