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Short Communication |
From the Department of Neuropathology,*
Faculty of
Medicine, University of Tokyo, Tokyo, and Core Research for Evolutional
Science and Technology (CREST),
Japan Science
and Technology Corporation, Kawaguchi, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tau, a microtubule (MT)-associated protein (MAP), binds to tubulin at three or four repeats of 31 or 32 amino acids each, called the MT-binding domain, located in the carboxyl half of tau. In adult human brain, six isoforms containing three or four repeats are generated by alternative mRNA splicing from a single gene. Six known missense mutations in tau in FTDP-17 are located within or close to the MT-binding domain, and the intronic mutations are in the 5' splice site of exon 10, and thought to be involved in the formation of a stem loop, resulting in an increased proportion of four-repeat tau.4-6 Regarding exonic mutations, it is tempting to speculate that these mutations in tau interfere with its interaction with tubulin and thus decrease its ability to promote tubulin assembly.3,4 Along this line of investigation, two groups recently reported independently that the exonic mutations cause significant defects in MT assembly in the cell-free system.8,9 However, acute depletion of tau was reported to have no effects on MT dynamics in cultured neurons10 and, further, tau-knockout mice showed remarkably subtle effects on neurogenesis and neuronal organization.11 These results suggest that the tau mutation effects on MT assembly found in the cell-free system may not always be problematic within the cells or in vivo. Accordingly, it is too early to speculate about the pathogenesis of FTDP-17 based on the results from the cell-free system alone. Furthermore, the effects of phosphorylation, which is known to affect the ability of tau to promote MT assembly, cannot be assessed by those cell-free experiments using Escherichia coli-produced tau.8,9 Thus, we have established Chinese hamster ovary (CHO) cells stably transfected with wild-type and exon-mutated tau cDNA to investigate the effects of the mutations within the cell.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following antibodies were used: rat monoclonal antibody to
tyrosinated 
-tubulin, YL1/2 (Harlan Sela-Lab, Hillcrest, UK); mouse
monoclonal antibody to -tubulin, N356 (Amersham Pharmacia Biotech,
Airlington Heights, IL); mouse monoclonal antibody to hamster
lysosome-associated membrane protein 2 (LAMP2), UH3 (Developmental
Studies Hybridoma Bank, Iowa City, IA); and mouse monoclonal and rabbit
polyclonal antibodies to tau, tau 1 (Roche Diagnostics,
Tokyo),12,13
AT8 (Innogenetics, Kapelanielaan,
Belgium),14
5E2,15
M4 and
C5,16,17
PHF 1,18
and AP422.19
MitoTracker Red was purchased from Molecular Probes, Inc. (Eugene, OR).
Construction for Expression Plasmids and Transfection
Unique EcoRI and BglII sites were introduced into the 5' and 3' noncoding regions, respectively, of four-repeat (0N4R) tau cDNAs without exons 2 and 3, using polymerase chain reaction. The EcoRI-BglII fragment was isolated from the amplified product and ligated with pCXN20 digested with the same endonucleases. The resultant recombinant plasmid, pCXN-4Rtau-wild-type, was used for further studies. To generate tau cDNA harboring mutations (G272V, P301L, V337M, and R406W, numbered according to the longest human tau isoform),21 the polymerase chain reaction/oligonucleotide primer-directed mutagenesis strategy was used. The entire nucleotide sequence was determined by the dideoxynucleotide termination method using a DNA sequencer (model 4000L; LI-Cor, Lincoln, NE). The resultant amplified fragment was digested with the appropriate endonucleases and then inserted into the same sites of pCXN. The derivatives were pCXN-4Rtau G272V, P301L, V337M, and R406W.
Vector alone, vectors containing 4R wild-type tau, and mutant tau were transfected into CHO cells using Lipofectamine (Life Technologies, Rockville, MD) according to the manufacturer's instructions. Clones that survived in G418 (Wako Pure Chemical, Tokyo; 0.4 mg/ml) were isolated and maintained at 37°C in 5% CO2 in Ham's F-12 medium (Life Technologies) supplemented with 10% fetal bovine serum containing G418 (0.1 mg/ml).
Biochemical Analysis
Cultured cells grown in 10-cm dishes were scraped into ice-cold Tris-buffered saline (50 mmol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl) with various protease inhibitors as described before.22 The homogenates were solubilized with sodium dodecyl sulfate and the protein concentration in each homogenate was determined by bicinchoninic acid protein assay (Pierce, Rockford, IL). For quantitative blotting, the homogenates containing the same protein amounts were spun at 5000 rpm for 5 minutes and resultant supernatants were treated by heat. The heat-stable fractions containing tau were concentrated with saturated ammonium sulfate.22 The alkaline phosphatase treatment was performed essentially as described elsewhere.22 Aliquots of protein were subjected to Western blotting with tau 1 or N356. The expression levels of tau and tubulin in each of the transfectants were quantitated using a GS700 Imaging Densitometer (Bio-Rad, Hercules, CA) using authentic tau or tubulin within a linear range, and particular cell sublines containing similar levels of tau (< mean ± 100%) were selected for the following studies.
Subcellular Fractionation and Isolation of Tau
Transfected cells from each confluent 15-cm culture dish were fractionated as previously described.23 Separation of polymerized tubulin and free tubulin was performed according to the protocol designed by Merrick et al.24 Tau, bound or unbound to MT in cultured cells, was extracted as described.25
Immunofluorescence Microscopy
Cells were fixed with cold methanol or 4% paraformaldehyde in MT-stabilizing buffer (80 mmol/L PIPES, pH 6.9, 1 mmol/L MgCl2, 1 mmol/L EGTA), followed by incubation in 0.3% Triton X-100 in phosphate-buffered saline for 5 minutes and incubation with antibodies essentially as described.26 For the secondary antibodies, donkey anti-mouse IgG conjugated with rhodamine or fluorescein and donkey anti-rat IgG conjugated with rhodamine were used (Jackson ImmunoResearch Lab, West Grove, PA). Anti-mouse and anti-rat antibodies were preabsorbed with rat and mouse nonimmune serum, respectively, to remove cross-reactivity. To visualize mitochondria, MitoTracker Red was added at a concentration of 300 nmol/L 30 minutes before fixation. In some experiments, cells were treated with 0.1 µg/ml Colcemid (Wako Pure Chemical) for 2 hours or 20 µmol/L cytochalasin B (Wako Pure Chemical) for 20 minutes. Labeled cells were examined under a Zeiss Axioskop microscope (Carl Zeiss Co., Tokyo).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, we examined by immunocytochemistry the intracellular
distribution of tau in transfected CHO cells that do not normally
express tau. When fixed with paraformaldehyde, the immunoreactivity of
tau 1 was distributed diffusely in the cytoplasm, suggesting that a
significant proportion of tau is unbound to MTs (Figure 1, a and b)
, an observation consistent
with a previous report.25
To see the tau bound to MTs, the
cells were fixed with cold methanol to wash away cytosolic
tau.26
With this fixation protocol, tau 1 immunoreactivity
was almost completely colocalized with MTs (Figure 1, c and d)
. In many
cells, MT bundles were observable after tau transfection (Figure 1, a–h)
27
but no distinct difference in the incidence and
localization of MT bundles was found among transfectants (Figure 1, c and e
-h). As expected, mock-transfected cells were completely negative
for tau (data not shown).
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. With the same treatment, a certain population of
wild-type tau, tauV337M, and R406W (hereafter referred to as WT,
V337M, and R406W, respectively) transfectants still showed MT staining
(Figure 1, n, q, and r)
, whereas such MT staining was very rare in
tauG272V and P301L (hereafter referred to as G272V and P301L,
respectively) transfectants (Figure 1, o and p)
. To quantitate these
results, the number of MT-positive cells per 1000 cells was counted
under a fluorescence microscope. The proportion of MT-positive cells
was 46% for WT, 48% for V337M, 37% for R406W, 14% for G272V, and
6.3% for P301L transfectants. Thus, G272V and P301L transfectants
showed significantly greater vulnerability to Colcemide than WT
transfectant (P < 0.01; Student's
t-test). This experiment was repeated four times with
similar results.
It was recently reported that overexpression of tau affects
kinesin-dependent trafficking of intracellular
organelles.29
To examine whether the mutations alter the
intracellular distribution of organelles, mitochondria and lysosomes
were labeled by MitoTracker Red and UH3 (anti-LAMP2 antibody),
respectively. In mock-transfected cells, both organelles were
distributed throughout the cytoplasm (Figure 1, i and k)
. In contrast,
in tau-transfected cells, mitochondria were confined to the perinuclear
area (Figure 1j)
, while lysosomes were scattered throughout the
cytoplasm (Figure 1l)
. These results were consistent with the
previously reported result,29
but again, no difference was
observed between wild-type and mutant transfectants.
It is well known that disruption of the subcortical microfilament network induces process formation in cultured cells.30 Because tau also binds to actin in several cell lines,31 we examined whether tau mutants had any effect on process formation. Treatment of the transfected CHO cells with cytochalasin B induced the outgrowth of MT-containing processes from the cell surface. Although mock-transfected cells tended to give off a greater number of processes than WT and mutant tau-transfected cells, there were no significant differences in the number and length of processes among the transfectants (data not shown).
Biochemical Analysis
Quantitation of polymerized tubulin in each transfectant provided
rather conflicting data. The expression levels of tubulin in tau
transfectants appeared to be slightly increased compared to that in
mock transfectants (Figures 2 and 3)
. The proportion of polymerized tubulin
was significantly decreased in WT, V337M, and R406W transfectants
compared to that in mock transfectants (P <
0.05, Mann-Whitney U test) (Figure 2)
. Furthermore, tubulin
in G272V and P301L transfectants was polymerized to a greater extent
than that in WT transfectants (P < 0.05,
Mann-Whitney U test). These results apparently contradict
the results on the ability of the mutants to promote MT assembly in the
cell-free system.8,9
Consistent with these results, tau
appeared to be much more abundant in the supernatant of each
transfectant homogenate (Figure 2
and legend). This probably agrees
with the above-described immunocytochemical observations of tau
transfectants showing diffuse cytoplasmic staining for tau when fixed
with paraformaldehyde.
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. The top band was stronger in
intensity than the lower two bands in WT, G272V, P301L, and
V337M-transfected cells. However, in R406W transfectants, the intensity
of the lowest band was much stronger than that of the upper two bands
(Figure 2)
. After dephosphorylation, the mobilities of tau in all
transfectants corresponded to that of the lowest band (Figure 2B)
.
Thus, the upper two bands and the lowest band represent differentially
phosphorylated and nonphosphorylated species of tau, respectively. The
three bands were probed with various phosphorylation-dependent and
phosphorylation-independent antibodies including tau
1,12,13
AT8,14
5E2,15
M4 and
C5,16,17
PHF 1,18
and AP422.19
Phosphorylation-dependent antibodies other than AT8 and AP422 (data not
shown) labeled exclusively the top band except in the R406W
transfectant. In this transfectant, only PHF 1 among
phosphorylation-dependent antibodies weakly labeled the top band on the
blot of its homogenate (Figure 3)
. PHF 1 recognizes phosphorylation at
both Ser 396 and Ser 404,18
suggesting that either site is
barely phosphorylated in R406W. To confirm this, C5, which is highly
specific for the phosphorylation only at Ser 396,17
was
used and found to be unable to label the band. This indicates that Ser
396 is barely phosphorylated in R406W. Presumably, a lesser extent of
the PHF 1 reactivity is due to lack of phosphorylation at Ser 396 and
possibly at other sites, including Ser 400 and Thr 403.19
The faint reactivity of M4 also indicates that R406W is less
phosphorylated at Thr 231 (Figure 3F)
.16
Altogether, in
the R406W transfectant phosphorylation of tau at Thr 231 and Ser 396 is
profoundly reduced compared to that in other transfectants. We also examined the subcellular localization of tau in the transfectants.23 The relative amounts of tau in cytosolic, membrane, and mitochondrial fractions were quantitated by Western blotting using tau 1. In all transfectants, the fraction with the greatest abundance of tau was the membrane fraction, followed by the cytosolic fraction (data not shown). The proportion of tau in the mitochondrial fraction was significantly greater in the V337M transfectant (P < 0.05, Mann-Whitney U test). Its proportion in the membrane fraction was significantly lower in the P301L transfectant (P < 0.05, Mann-Whitney U test), whereas that in cytosolic fraction was significantly greater in the P301L transfectant (P < 0.05, Mann-Whitney U test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we sought to examine the effects of the mutations
on MT assembly in cultured cells, because there may be significant
differences between cell-free and intracellular conditions. In fact,
transfection with cDNA containing the MT-binding domain alone which
worked in the cell-free system had no visible effects in cultured
cells.26
Small regions on both sides of MT-binding domain
of tau were shown to be essential for its MT localization in cultured
cells.26
For this reason, we established stable cell lines
expressing four-repeat (0N4R) tau without and with four missense
mutations and examined the effects of these mutations within cells. In
both wild-type and mutant transfectants, the proportion of polymerized
tubulin was decreased compared to that in mock transfectants. In
parallel with this, a much greater proportion of tau was recovered in
the supernatant containing nonpolymerized tubulin (Figure 2)
. We do not
know the exact reason for this, but possible explanations include (i)
increased levels of tubulin in the tau transfectants may reduce an
apparent effect of tau, (ii) MTs in CHO cells may be almost saturated
with MT-associated proteins other than tau so that only a fraction of
tau can have an effect on MT assembly and stabilization, and (iii) it
is possible that tau may interact with unknown proteins other than
tubulin (see below), which may reduce the interaction between tau and
tubulin. Thus, it is possible that the presence of tau (and tubulin)
does not necessarily lead to MT assembly within the cell, an
observation that contrasts with observations of the cell-free
system.
The transfectants used for the present analyses exhibited similar
levels of expression of tau (the maximal difference was within twofold;
data not shown) and of tubulin (Figure 3)
. Thus, any differences in the
phenotype of CHO cells can probably be ascribed to the effects of
mutation, although we cannot completely exclude the possibility that
some of the effects of the mutants are caused by inherent differences
in the expression levels of tau and tubulin. With this
reservation, we carefully searched for any differences between
wild-type and mutant tau and failed to find any effects consistently
attributable to mutant tau in CHO cells. It is possible that loss of
function of mutant tau is compensated by other MT-associated proteins
in CHO cells that do not normally express tau. However, a fraction of
transfected tau was colocalized with MTs and led to MT bundling (Figure 1)
. Furthermore, wild-type and some mutant tau appeared to stabilize
MTs to a greater extent against Colcemide when compared with the mock
transfectant (Figure 1)
. Thus, it is likely that a fraction of
transfected tau has a discernible effect on the stabilization of MT in
transfected CHO cells. The results on MT assembly in the cell-free
system should reflect MT nucleation and growth,8,9
but not
saturation levels, and stabilization of MTs, which could be a more
important role of tau within the cell32
and presumably
in vivo. Thus, our observations suggest that the reported
partial loss of function of mutant tau8,9
may not be
critical for causing FTDP-17.
The Colcemid treatment allowed us to differentiate the three mutations in the MT-binding domain into two groups: G272V and P301L in one group and V337M in the other. G272V and P301L are located in the characteristic MT-binding motif, PGGG, whereas V337M is located two residues downstream of the carboxyl terminus of the third repeat. The neuropathological features reported in the affected brains harboring these mutations are quite different.2,33 These findings may raise the possibility that the different pathologies caused by mutations within or outside the PGGG may be related to the MT stability following Colcemid treatment. However, biochemical quantitation of polymerized tubulin after Colcemid treatment did not support the view that G272V and P301L transfectants are more susceptible to Colcemid than V337M or WT transfectants (data not shown). This apparent contradiction may be related to the facts that immunofluorescence analysis here focused only on the MT-bound tau, not total tau (see above) and that only a minor proportion of tubulin is polymerized in the tau transfectants. The MTs remaining after Colcemid treatment could represent a very small proportion of tubulin, and thus may not become apparent by biochemical quantitation.
One mutation, R406W, is located close to key residues for phosphorylation, Ser 396, 400, and 404, and Thr 403, all of which are hyperphosphorylated in PHF-tau.19 Thus, one can reasonably speculate that this mutation could have a profound effect on phosphorylation on these sites.3,4 These sites may tend to be more easily phosphorylated and phosphorylated species may destabilize MTs, leading to neuronal degeneration. In fact, the phosphorylation at Ser 396 was previously shown to substantially decrease the affinity of tau for MTs.25 In view of this, it is surprising to note that in the R406W transfectant, tau cannot be phosphorylated at this site. The greater resistance of the R406W transfectant to Colcemid may be also explained by the lesser extent of the phosphorylation at Ser 396, because S396A-transfected CHO cells showed greater resistance to nocodazole-induced MT depolymerization than cells expressing wild-type tau.25 Indeed, in brains affected by the R406W mutation, tau filaments are formed and Ser 396 in the tau comprising those filaments is hyperphosphorylated.34 Interestingly, this mutation also leads to decreased phosphorylation at Thr 231, which is also abnormally hyperphosphorylated in tau filaments in affected brains (L Reed, personal communication).34 These results could be of particular importance for considering the significance of hyperphosphorylation of tau in filaments. Because in any transfected cell there is abundant unpolymerized tubulin in the cytoplasm, cytosolic tau is likely to bind to free (monomeric and oligomeric) tubulin; tau in the cytoplasm exists largely in the tubulin-bound form. It is possible that this tubulin-tau complex is the substrate for various protein kinases involved in tau phosphorylation. In the case of R406W, Ser 396 are not phosphorylated to any significant extent, possibly due to an aberrant conformation under normal conditions. Thus, for the two aforementioned sites of R406W to be fully phosphorylated as seen in tau aggregates in the brain with the R406W mutation, some profound derangement of MT including MT disruption in the cell would be required. The hyperphosphorylation at these sites could occur in MT-unbound tau instead of MT-bound tau, or in tubulin-stripped tau instead of tubulin-bound tau. Assuming that the neurodegeneration in FTDP-17 is due to an unknown function of mutant tau (see below), possibly due to its gain of function, the above result suggests that such hyperphosphorylation occurs in degenerating neurites and neuronal perikarya where MTs are depolymerized or tubulin is being lost. Tau could be left behind because of greater resistance to proteases and greater aggregation potential.35 This points to the possibility that tau aggregation by itself is not the cause but a consequence of neuronal degeneration.
Tau was initially isolated as a factor that stimulated MT assembly in
the cell-free system,36
but now it is known that this
protein appears to play many other roles by interacting with
actin,37
ßPP,38
PP2A,39
PP1,40
phospholipase C
,41
presenilin
1,42
and src-family nonreceptor tyrosine
kinase43
(for review see Ref. 44
). As shown above, tau is
not only a cytosolic protein but also exists in other compartments
including the nucleolus,45
nucleus,46
and
plasma membrane.23
Thus, tau probably plays important
roles other than MT assembly and stabilization in vivo, and
much more attention should be paid to such functions of tau. This
assumption may be consistent with a recent observation that N279K and
S305N mutations do not affect the ability to promote MT assembly, but
have potential effects through alteration of the splicing of exon
10.47
Finally, it should be noted that the role of
MT-associated proteins in MT dynamics depends on the cell
type.48
These cell type-specific roles of MT-associated
proteins may explain why the present study was unable to uncover any
abnormal phenotype shared by all mutant tau transfectants. In this
context, studies using neuronal cells and transgenic animals are
essential to an understanding of the significance of tau mutations
leading to tau aggregation and neuronal loss in FTDP-17.
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Acknowledgements |
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Footnotes |
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Accepted for publication March 10, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is widely expressed in rat tissues. J Neurochem 1996, 67:1235-1244[Medline]
in situ. NeuroReport 1998, 9:67-71[Medline]
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