(Received for publication, April 19, 1995)
From the
Neurofibrillary tangles, which form in certain degenerating neurons in the brains of patients with Alzheimer's disease, are amassed from filaments having a straight or paired helical morphology. Solubilization of these filaments reveals that they are composed of the microtubule-associated protein tau. It has not previously been shown, however, that tau will assemble to form filaments of similar morphology under conditions representative of the intracellular environment. We have succeeded in forming such filaments using tau purified from porcine or rat microtubules. The filaments are relatively straight with narrowing at irregular intervals, and are about 10 nm wide, a morphology similar to that of straight filaments seen in Alzheimer's disease neurofibrillary tangles. At tau concentrations of 1-10 µM, in vitro assembly occurs at physiological pH, ionic strength, temperature, and reducing potential, and each one of these factors modulates the reaction. Assembly is judged to be only slowly reversible by the exponential rather than normal distribution of filament lengths, and by the limited disassembly observed under conditions which inhibit polymerization. Tau purified directly from whole brain tissue rather than from microtubules does not polymerize under conditions described in this report.
The intracellular fibrillar pathology of Alzheimer's
disease is characterized by the presence of filaments having a straight
or paired helical morphology (Kidd, 1963; Yagishita et al.,
1981). These filaments accumulate in both the somal neurofibrillary
tangles (NFT) ()and the dystrophic neuropil threads (Braak et al., 1986; Kowall and Kosik, 1987). The formation of NFT
and dystrophic neurites are spatially correlated (Probst et
al., 1989; Yamaguchi et al., 1990), and both lesions are
highly correlated with the severity of dementia (McKee et al.,
1991). Filamentous inclusions of this type are also seen in
Down's syndrome (Wisniewski et al., 1985), Guamanian
Parkinsonism-dementia (Hirano et al., 1968), and other disease
states (Wisniewski et al., 1979). In progressive supranuclear
palsy, NFT are composed primarily of filaments possessing the straight,
unpaired morphology (Tellez-Nagel and Wisniewski, 1973; Bugiani et
al., 1979). Although the death of polymer-laden neurons is
evidenced by the presence of insoluble tangle remnants in the
extracellular space, it is not known whether polymer masses disrupt
neuronal function sufficiently to induce degeneration, or whether they
merely form preferentially within neurons already involved in the
necrotic process.
Straight filaments (SF) and paired helical filaments (PHF) form under similar conditions, as evidenced by their co-existence within individual NFT (Perry et al., 1987). SF share epitopes with PHF and copurify with PHF in protocols that exploit their resistance to SDS or protease treatments (Perry et al., 1987; Crowther, 1991). In addition, there are several reports of transitional forms of fibrils possessing stretches of straight, then paired helical morphology continuous within a single filament (Wischik et al., 1985; Perry et al., 1987; Papasozomenos, 1989; Crowther, 1991). These findings and others have led to speculation that SF and PHF are formed by similar mechanisms of assembly (Perry et al., 1987; Crowther, 1991; Wille et al., 1992).
The only known structural constituent of the PHF is the microtubule-associated protein tau (for a review of the normal biology of tau, see Lee(1990)). The presence of tau proteins has been demonstrated both by immunochemical means (Grundke-Iqbal et al., 1986; Kosik et al., 1986) and by sequencing of peptides extracted from PHF (Wischik et al., 1988; Kondo et al., 1988). Tau extracted from PHF contains more phosphorylated residues than tau isolated from normal brain (Hasegawa et al., 1992; Ksiezak-Reding et al., 1992), and these phosphorylations are frequently invoked as being involved in the polymerization process.
Tau purified directly from brain or from
brain microtubules (MT) has been reported to form a variety of polymers
resembling SF or PHF. Dialysis of porcine MT tau for several days
against 6-8 M urea produced polymers ranging in width
from 5 to 35 nm, which included a subset resembling PHF (Montejo de
Garcini et al., 1986; Montejo de Garcini and Avila, 1987). The
effects of urea were attributed to deamination of glutamine residues or
carbamylation of lysine residues, although producing these
modifications by enzymatic or chemical means did not fully reproduce
the effects of urea treatment alone. Urea-treated tau was reported to
assemble independent of NaCl concentration in the range of 0.1-1 M. Using tau purified directly from bovine whole brain, 10-nm
filaments were formed in the presence of the cross-linking enzyme,
transglutaminase, under conditions optimized for enzymatic activity
(Dudek and Johnson, 1993). It is unlikely that this enzyme is required
for tau polymerization in vivo, however, since monomeric tau
can be readily solubilized from isolated PHF (Greenberg and Davies,
1990; Lee et al., 1991). Polymer formation has also been
demonstrated using bacterially expressed human recombinant tau. Two
groups using deletion constructs roughly equivalent to the MT binding
domain of tau and similar acidic conditions produced several polymer
species, which included a subset possessing the twisted morphology of
PHF (Wille et al., 1992; Crowther et al., 1992).
Full-length tau constructs did not assemble under these conditions.
More recently, however, using conditions of neutral pH and high ionic
strength (1.25 M
CHCO
-K
), full-length tau
constructs were observed to form filaments, some of which resembled PHF
(Crowther et al., 1994).
The establishment of causal relationships between the assembly of tau into SF and PHF, and potential modulating factors such as phosphorylation or other enzymatic or chemical treatments, would benefit from an in vitro assembly system in which these polymers can be demonstrated to form under physiologically relevant conditions. We have defined the conditions in which tau purified from rat or porcine MT will assemble into a homogeneous population of filaments resembling SF. Although kinetically a relatively slow process, in vitro filament formation is observed under essentially physiological conditions.
Fresh bovine brains were obtained from John Morrell Meat Packing, Montgomery, AL. Fresh porcine brains were obtained from Bryan Meat Packing, Westpoint, MS. Human brain was provided by Dr. Richard Powers of the Brain Resource Center, University of Alabama at Birmingham. Sprague-Dawley rats were obtained from Charles River Laboratories, Wilmington, MA, and killed by decapitation. Purified NFT (Iqbal et al., 1984) were provided by Dr. Khalid Iqbal, Institute for Basic Research in Developmental Disabilities, Staten Island, NY. Neurofilament antibodies (Amersham Corp.) and protein were provided by Dr. Robert Goldman, Northwestern University Medical School, Chicago, IL.
Tau was isolated
using protocols similar to those published by others (Sandoval and
Weber, 1980; Johnson et al., 1989), exploiting the
protein's stability to heat treatment (Cleveland et al.,
1977a) and solubility in perchloric acid (Lindwall and Cole, 1984). For
isolation from microtubules, pellets were resuspended in cycling buffer
(100 mM PIPES, 1 mM EGTA, 1 mM
MgCl, pH 6.9) supplemented with 0.8 M NaCl and 2
mM DTT, stirred on ice for 30 min, boiled for 10 min, stirred
on ice for 30 min, and centrifuged at 100,000
g for 45
min. Supernatants were concentrated over an Amicon YM10 ultrafiltration
membrane, and loaded on a Bio-Gel A-1.5 sieve column (32
430
mm, run at 15 ml/h), equilibrated with buffer A (20 mM MES, 80
mM NaCl, 2 mM EGTA, 1 mM MgCl
,
0.1 mM EDTA, pH 6.8) supplemented with 0.8 M NaCl and
2 mM DTT (buffer A+). Fractions containing tau were
brought to 2.5% perchloric acid, stirred on ice for 30 min, and
centrifuged at 100,000
g for 30 min. Supernatants were
dialyzed against buffer A and concentrated by ultrafiltration. Residual
DTT was estimated to be
0.2 µM. All procedures except
boiling were carried out at 4 °C.
For isolation of tau directly
from whole brain, frozen tissue was thawed and homogenized (1:1, w/v)
in a Waring blender in buffer A+ supplemented with an additional
dry weight of NaCl sufficient to bring the homogenate to 0.8 M NaCl. Following centrifugation at 150,000 g for
45 min, the supernatant was boiled 10 min, iced 30 min, and centrifuged
as above. The supernatants were then brought to 60% saturation with
(NH
)
SO
, stirred on ice 45 min, and
centrifuged at 100,000
g for 30 min. Pellets were
resuspended in a total of 50 ml of buffer A+, dialyzed against the
same solution, concentrated by ultrafiltration, and loaded on the sieve
column. Procedures described above for isolation of tau from
microtubules were then followed.
Figure 6: Filament lengths display an exponential distribution. P14 rat tau was incubated in 10 mM DTT at 22 °C (A-E) or 37 °C (F-J) for the indicated number of hours (t). Histograms were generated using filament measurements obtained as described under ``Materials and Methods.'' Average filament length (x) and sample size (n) are also given for each data set. Bin width is 200 nm.
Figure 7:
Polymerization of tau filaments is
dependent on ionic strength. Porcine tau in buffer A was diluted 1:1
into neutral 50 mM Tris, 20 mM DTT supplemented with
a variable amount of KCl. Samples were incubated for 24 h at 37 °C
and then supplemented with another 500 mM KCl. One aliquot was
taken immediately for EM processing (opencircles),
and another after an additional 24-h incubation at 37 °C (filledcircles). Random fields were digitized, after
which filaments were measured and their lengths were summed. The
average total polymer length/field ± S.E. is plotted as a
function of the initial cation concentration, n
10.
Figure 9: Filament lengths are a function of the reducing potential. A-E, porcine MT tau was incubated 24 h with the indicated concentration of DTT. Samples were deposited on grids, and filaments from 10 randomly chosen fields were digitized and measured. Bin width is 300 nm. F, the average length of each filament population is plotted as a function of DTT concentration.
Figure 1: Polymerization of rat and porcine tau protein. MT tau from P14 rat (A, F, and G), P10 rat (B), adult rat (C and D) or adult pig (E and H-K) was polymerized as described under ``Materials and Methods'' and stained with uranyl acetate. In addition to dispersed preparations, typical patterns of non-filamentous (B) and inter-filamentous (C) aggregation are shown. Taxol-stabilized MT (arrowheads) were co-precipitated on some grids for size reference. Bar equals 500 nm (A-E) or 100 nm (F-K).
For colloidal gold labeling, following
deposition on grids and an HO rinse, grids were inverted
for 1 h on a 10-µl drop of the primary antibody (Tau-2;
Papasozomenos and Binder, 1987) diluted in borate saline (0.1 M H
BO
, 25 mM
Na
B
O
, 75 mM NaCl). Grids
were then rinsed with borate saline, inverted on a drop of secondary
antibody (Ted Pella, 10 nm colloidal gold conjugated goat anti-mouse,
diluted 1/125), rinsed with borate saline supplemented with 1.5 M NaCl, and stained with 2% uranyl acetate.
Polymerization of MT tau was accomplished by diluting the
purified protein into neutral Tris or MES buffers in which sulfhydryl
reactivity was limited by the presence of reducing agents. Polymers
formed under a variety of conditions are shown in Fig. 1.
Polymerization was demonstrated using tau from juvenile rat (indicated
by postnatal age), adult rat, and adult pig, at protein concentrations
of 1.6-6.5 µM. Incubations were done at 37 °C
for 5-26 h, using 5-25 mM ME or 2-20
mM DTT. The filaments formed resemble the SF known to reside
within the NFT, in the sense that they are non-helical and unpaired.
The obvious flexibility of some filaments (Fig. 1D),
however, makes them straight in a strictly non-Euclidean sense, and
distinguishes them from the rather rigid appearing PHF (Wisniewski et al., 1984) and SF (Crowther, 1991). The tau polymers at
their widest extent have an average width of 10.5 nm, with a measured
range of 6.5-13 nm. Filaments narrow for variable distances at
irregular intervals (Fig. 1, H and J) to about
50% of their widest extent. These narrowings likely represent the
crossover points of a slightly twisted filament. SF isolated from NFT
also show a modulation of width interpreted as resulting from a
twisting of the long axis (Crowther, 1991). The relatively large range
of filament diameters observed is believed to result from this variable
twisting combined with the final disposition of filaments on the
support film, and not from multiple filament morphologies. Differences
in staining intensity, which may create the appearance of multiple
filament populations within a single field (Fig. 1A),
are commonly observed. These are due to the unequal contributions of
positive and negative staining across the grid surface, and again, do
not indicate the presence of multiple filament morphologies.
Two types of aggregation often seen in preparations of polymerized tau are also shown in Fig. 1. The first is most common in the juvenile tau samples and appears to involve the precipitation of nonfilamentous tau onto tau filaments (Fig. 1B). Because of the electron opaqueness of these aggregates, we cannot rule out the possibility that they are composed of highly compacted short filaments. They are, however, usually associated with a single discernible tau filament. The appearance of such aggregates is usually associated with an obvious decrease in the density of filaments seen by electron microscopy, and confounds attempts to quantify polymerization by centrifugal separation of soluble and filamentous tau. Bundling of tau filaments is also occasionally observed (Fig. 1C). Both forms of aggregation appeared to be reduced when the Tris assembly buffer was replaced with borate saline (data not shown), although this appears to reduce polymer yields.
To date, we have tested 15 tau isolates for their ability to form filaments. Although all of the tau preparations purified from twice-cycled MT (11) exhibit the ability to assemble, none of the preparations purified directly from whole brain extracts (4) are assembly competent (see ``Materials and Methods'' for differences in purification protocols). The assembly incompetent tau included isolates from bovine, porcine, and human brain. Tau purified by both methods is presumed to have the same primary structure, but is likely to exist in different states of phosphorylation owing to the activity of phospatases and kinases present during MT cycling (Tsuyama et al., 1986; Burns, 1991). Differences in levels of specific post-translational modifications may be involved in stimulating or suppressing polymerization.
The morphology of in vitro assembled tau polymers was compared to that of Alzheimer's SF and PHF (Fig. 2). NFT were isolated using an SDS extraction protocol (Iqbal et al., 1984), and processed for electron microscopy in the same manner as the tau filaments. Most of the filaments found in these preparations exhibited the typical PHF morphology (Fig. 2A). Occasional SF were observed, and, rarely, a NFT fragment composed almost exclusively of SF was observed (Fig. 2B). A comparison of SF and tau filaments (Fig. 2C) revealed two filament populations, which were similar in width and which presented a smooth surface lacking detectable substructure by this staining method. The PHF at their widest extent were significantly wider than the SF or the in vitro assembled tau filaments. The SF found in purified NFT preparations were apparently more rigid than tau filaments (Fig. 1D) and often appeared to be broken when forced by inter-filament contacts to attempt significant bending. This may result from SF containing a greater number of phosphorylated residues, analogous to the increased rigidity of paracrystals formed from phosphorylated tau (Hagestedt et al., 1989).
Figure 2: Comparison of tau polymers assembled in vivo and in vitro. Purified NFT (A and B) and tau filaments (C) were stained with 2% uranyl acetate. Examples of both PHF (A) and SF (B) are shown. Bar equals 0.1 µm.
Owing to the fact that our polymers are also similar in size to neurofilaments, and that neurofilaments are a known contaminant of the cycled MT from which we purify our assembly competent tau (Berkowitz et al., 1977), we tested our tau isolates for possible contamination by neurofilament proteins. A Coomassie stain of two typical tau preparations separated by SDS-PAGE revealed dye binding only to the tau bands (Fig. 3, A and B). A Western blot of an identical gel using a monoclonal antibody directed against neurofilament light chain showed binding to 40 ng of a partially purified neurofilament preparation (Fig. 3E; binding was also detected at 10 ng), but no binding to the tau proteins loaded at 5-10 µg/lane (Fig. 3, C and D). The maximum potential neurofilament contamination was calculated to be <0.1%.
Figure 3: Purification of tau to apparent homogeneity. A-D, microtubule tau from P14 rat (5 µg, A and C) or adult rat (10 µg, B and D) was separated on 4-20% gels and stained with Coomassie Blue (A and B) or blotted with a monoclonal antibody that binds the 68-kDa neurofilament protein (C and D). Neurofilament protein was also blotted (40 ng, E) or stained with Coomassie Blue (15 µg, F). Molecular size markers (in descending order: 208, 101, 71, 44, 29, and 18 kDa) are indicated to the left, and the neurofilament triplet proteins on the right. Lanes C-E were excised from the same blot.
To further confirm that in vitro assembled filaments were composed of tau, EM localization was performed using a tau monoclonal antibody in conjunction with gold-conjugated secondary antibodies. Gold particles were specifically associated with filaments when filaments were preincubated with the Tau-2 monoclonal antibody (Fig. 4, A and B), but not when the primary antibody was omitted (Fig. 4, C and D). Treatment of filaments with 0.5 M KCl to remove proteins bound nonspecifically to the surface of filaments did not effect labeling (Fig. 4, E and F).
Figure 4: Antibody labeling of tau polymers. Porcine tau was assembled overnight, and filaments were either deposited directly on grids (A-D) or incubated in 500 mM KCl for 30 min at 37 °C prior to being deposited on grids (E and F). Grids were then incubated with the tau monoclonal antibody, Tau-2 (A, B, E, and F), or control buffer (C and D), followed by incubation with a secondary antibody conjugated to 10 nm colloidal gold. Grids were then stained with uranyl acetate. Two examples of gold labeling for each condition are shown. Bar equals 0.2 µm.
Analysis of filament assembly revealed that the extent of polymerization was dependent on incubation times and temperatures (Fig. 5). Increasing incubation times resulted in the presence of longer filaments on the grid surface. When tau was incubated at 37 °C (Fig. 5, F-J), the filaments formed appeared to be greater in length and number than those formed at 22 °C (Fig. 5, A-E). In this preparation of P14 rat tau, no polymerization was evident after a 4-h incubation at 4 °C. In subsequent experiments with adult rat tau, however, some polymerization was observed at 4 °C (data not shown).
Figure 5: Filament length is dependent on time and temperature. Tau purified from P14 rat MT was incubated in 10 mM DTT at 22 °C (A-E) or 37 °C (F-J) for 15 min (A and F), 30 min (B and G), 60 min (C and H), 120 min (D and I), or 240 min (E and J). Samples were processed for EM, and representative fields were recorded. Bar equals 1.5 µm.
Electron micrographs similar to those seen in Fig. 5were digitized so that filament lengths could be measured (Fig. 6). The histograms generated confirm that the average filament length increases with time, and, at any given time, filaments in the 37 °C sample are longer than those assembled at 22 °C. The data also show that filaments display an exponential length distribution, rather than the Gaussian distribution that is usually seen in microtubule populations (Symmons and Burns, 1991). This type of distribution would be consistent with a filament population that was adding nucleation sites at a constant rate and exhibiting limited subunit dissociation.
The dependence of filament assembly on ionic strength was also examined. As neutral solutions (25 mM Tris, 40 mM NaCl) were supplemented with 0-75 mM KCl, the filament mass subsequently found on grid surfaces was markedly decreased (Fig. 7). All samples were supplemented with 500 mM KCl prior to their deposition on grids, so this decrease is not due to differential adherence to the grid surface, which could have otherwise resulted from the variable salt content of the samples. Filaments were observed at low density when KCl was added at 100 mM (Tris + Na + K = 165 mM), but complete inhibition was routinely observed at higher salt concentrations (data not shown).
With respect to filament depolymerization, increasing KCl concentrations well above assembly-inhibiting concentrations caused only limited filament disassembly. Filaments were still observed when samples preassembled for 24 h were raised to over 500 mM KCl and incubated another 24 h (Fig. 7). This suggests that assembled filaments display only limited subunit dissociation, consistent with the data obtained on filament length distributions (Fig. 6).
The effect of pH on
tau polymerization is shown in Table 1. Tau from P14 and adult
rat was incubated overnight in 20 mM ME buffered at
variable pH, then assayed for filament formation by electron
microscopy. Assembly occurred over the broad pH range between 6 and 11,
but was largely inhibited at pH 5.6 and completely inhibited at lower
pH. Samples that were incubated overnight at inhibitory pH and then
raised to neutral pH exhibited normal assembly (data not shown).
In
our earliest attempts at in vitro polymerization of P14 rat
tau, it was readily apparent that reducing agents must be included in
the incubation buffer if filaments of sufficient length to be
recognized as such were to be formed. The relation between ME
concentration and the length of filaments assembled from P14 tau is
shown qualitatively in Fig. 8. As
ME concentration was
lowered from 0.1 M (Fig. 8B) to 0.1 mM (Fig. 8E), a decrease in maximum filament length
was observed. At low
ME concentrations, a concomitant increase in
particles smaller than those recognized as filaments was often observed (Fig. 8, D and E). These may represent minimal
length polymers. Similar data were produced using porcine tau and DTT
as the reducing agent (Fig. 9). Although in this experiment the
average filament length clearly decreased as the concentration of DTT
was lowered (Fig. 9F), the length of filaments observed
at the lowest level of reducing agent was more variable than that seen
in other experiments (Fig. 8), (
)with filaments as
long as 2.4 µm recorded. This variation reflected the variable
extent to which different preparations of MT tau were observed to
polymerize, with those preparations that produced the highest level of
polymer typically exhibiting a more uniformly short population of
filaments at low concentrations of reducing agents. Although high
concentrations of reducing agents were used in many experiments to
maximize the assembly of long filaments, it should be emphasized that
the range of reducing potentials represented in Fig. 9would
clearly encompass values expected to occur in the cytoplasm.
Physiological concentrations of glutathione (1-10 mM,
Hwang et al., 1992), the tripeptide that constitutes
most of the redox buffering capacity of the cytoplasm, also promoted
the assembly of MT tau purified from P11 rat, adult rat, and adult
porcine brain (data not shown).
Figure 8:
Tau
polymerization is dependent on reducing potential. P14 rat tau was
incubated overnight at pH 7.2 with ME added to 1.0 M (A), 0.1 M (B), 10 mM (C), 1.0 mM (D), 0.1 mM (E), or 0.0 mM (F). Samples were
processed for EM, and representative fields are shown. Bar equals 1 µm.
We have succeeded in assembling a homogeneous population of
tau filaments that appear to be morphologically related to the SF seen
in Alzheimer's disease. It has not previously been demonstrated
that tau will form polymers of this nature under conditions typical of
the intracellular environment. Previous studies describing the assembly
of tau filaments possessing straight or paired helical morphologies
(widths ranging from 10 to 25 nm) have depended on the use of
non-physiological conditions. Assembly of bovine or porcine tau was
previously achieved only after the chemical (Montejo de Garcini et
al., 1986) or enzymatic (Dudek and Johnson, 1993) modification of
the protein. Assembly of recombinant tau constructs containing either
the complete or partial tau sequence relied on either high salt
concentrations (1.25 M
CHCO
-K
; Crowther et
al., 1994) or an acidic pH (Wille et al., 1992, Crowther et al., 1992). None of these previous studies utilized
reducing agents in their assembly reactions. In this paper we
demonstrate that in a reducing environment, the assembly of 10-nm tau
filaments occurs when the variables of temperature, pH, and ionic
strength are adjusted to physiological values. Of these variables, only
ionic strength is limiting at physiological values, with optimal
assembly occurring at lower salt concentrations. Although filament
densities observed at physiological ionic strength were low,
significant polymer mass would likely accumulate in situ on a
longer time scale associated with the pathogenesis of the disease
state.
We believe that tau polymers formed in vitro, as well as SF formed in vivo, are ribbon-like in the sense that in cross-section their width is about twice their thickness, and that their measured profile depends on the extent to which they are lying either flat or on edge. Consistent with this interpretation is the two-fold difference in the range of widths measured for tau filaments. An example of a filament probably laying partially on edge is seen in Fig. 1J, where the central segment is very narrow but the ends are seen to flare to more typical dimensions. This interpretation of the filament morphology implies that the actual maximun width of these tau polymers is closer to the range maximum (13 nm) than the average measured width (10.5 nm). Although values of 15 nm reported for the width of SF in thin sectioned tissue are somewhat greater (Metuzals et al., 1981;Yagishita et al., 1981), overestimates because of the deposition of positive stain may result from this technique (Ruben et al., 1993).
Because of their similar dimensions, the possibility that the polymers assembled from our tau preparations were actually neurofilaments was addressed. Coomassie-stained gels of representative tau samples revealed no neurofilament or other protein contaminants, and Western blots of the same samples revealed no proteins cross-reactive with an anti-neurofilament protein antibody. The polymers formed were heavily labeled by an anti-tau antibody, even after incubating filaments in high salt to remove peripherally bound proteins. There were also differences between assembly conditions described in this paper and those previously reported for the assembly of neurofilaments. The assembly of tau filaments was favored at low ionic strength and inhibited at high ionic strength, while neurofilaments are assembled at high ionic strength (Geisler and Weber, 1981) and disassembled at low ionic strength (Hisanaga and Hirokawa, 1988). In addition, lowering the temperature or raising the pH of buffers used for filament assembly had no effect on the morphology of tau filaments (data not shown), but resulted in the formation of neurofilaments exhibiting abnormal morphologies (Aebi et al., 1988).
Following the time course of polymerization at different temperatures reveals three points of interest. First is the observation that all filament populations exhibited an exponential distribution of filament lengths, a fact confirmed by the length distributions generated over a range of DTT concentrations. This type of distribution is in contrast to the Gaussian distributions observed in equilibrated microtubule populations (Symmons and Burns, 1991) and is consistent with a filament population that exhibits limited subunit dissociation and adds nucleation sites at a constant rate. This interpretation is supported by the observation that tau filaments incubated at salt concentrations sufficient to inhibit assembly displayed only limited disassembly, again suggesting a relatively slow rate of subunit dissociation. A second point of interest is that the polymer mass formed increases as the temperature increases. This parallels the results of circular dichroism studies, which demonstrated that the secondary structure content of tau also increased as a function of temperature (Ruben et al., 1991). It is likely, therefore, that prior to polymerization the tau molecule assumes a more highly ordered conformation. The third point is that tau filaments appear to lengthen with time while maintaining constant radial dimensions. This suggests that filaments are elongating by the endwise addition of subunits, as opposed to the lateral condensation of preformed protofilaments. This was not evident in previous studies, which have all examined polymers from single time points.
A role for
tau dimers in the assembly of PHF-like polymers has previously been
suggested (Wille et al., 1992). Treatment of tau constructs
with phenylenedimaleimide to induce non-reducible cross-linking of
cysteine residues resulted in the formation of tau dimers as
demonstrated by SDS-PAGE, and these dimers assembled to form filaments
identical to those formed by untreated tau (Wille et al.,
1992). Our observation that filament lengths decrease as ME or DTT
concentration decreases suggests that tau dimers forming in a less
reducing environment may inhibit the polymerization process. It has
previously been shown in MT populations, however, that a factor that
stimulates assembly (microtubule-associated proteins) reduces the
average MT length (Sloboda et al., 1976). This illustrates the
inverse relationship that can exist between assembly rate and filament
length and points out that our data are open to more than one
interpretation, since we measured filament lengths as opposed to total
polymer mass. Although disulfide bonds resistant to cytoplasmic
reduction are found in globular proteins, they are not expected in a
protein like tau, which is believed to exist in an extended
conformation with limited secondary structure (Cleveland et
al., 1977b, Hirokawa et al., 1988). Since the region of
tau that contains the cysteine residues is known to be a relatively
hydrophobic part of the molecule (Ruben et al., 1991),
however, it is possible that conformational changes associated with
dimerization and/or polymerization trap the relevant cysteines in a
central hydrophobic domain not easily accessed by reducing agents.
The involvement of disulfide bonds may also be indicated by the pH
dependence of assembly (Table 1). The inhibition of assembly seen
below pH 6.0 could be due to decreased sulfhydryl reactivity, although
in this pH range effects due to histidine protonation cannot be ruled
out. It is interesting to note that the assembly of tau deletion
mutants was also observed to be pH-dependent. Under non-reducing
conditions, constructs encompassing most of the MT binding domain were
not observed to assemble at neutral pH, but did form filaments at pH
5.0-5.5 (Wille et al., 1992) or pH 4.5-5.0
(Crowther et al., 1992). We have also observed that under
non-reducing conditions, tau preparations that do not form filaments of
significant length at neutral pH (Fig. 8F) will form
long filaments at pH 5.5 (data not shown). Taken together these data
suggest that, although a sufficiently low pH will completely block
polymerization, under conditions more conducive to assembly the effects
of pH and reducing agent on disulfide stability are additive, and
increasing either [ME] or [H
]
will increase the average filament length.
Although tau purified from MT was observed to form filaments using our assembly conditions, tau purified from whole brain appeared assembly-incompetent. Another study, however, was able to demonstrate the presence of 10-nm filaments in samples of whole brain tau incubated with transglutaminase, an enzyme that catalyzes intermolecular cross-links between glutamic acid and lysine residues (Dudek and Johnson, 1993). The formation of filaments morphologically similar to those shown in our study suggests that whole brain tau is capable of associating in a manner similar to that which precedes the polymerization of MT tau. However, in the absence of the cross-linking enzyme, it appears likely that in the case of whole brain tau, this association is reversible. This implies that differences in the post-translational modification of tau purified by these two methods can directly affect the stability of cohesive tau interactions, and that transglutaminase can induce assembly independent of these modifications.
Phosphorylation affects many of the structural and functional properties of tau. Given that we are unable to isolate normal tau in a phosphorylation state identical to that of tau that has been incorporated into PHF and SF, it is not surprising that we are unable to exactly duplicate these morphologies. Tau filaments assembled in vitro did not resemble PHF and exhibited greater flexibility than SF. Specific phospates, which might allow for the lateral association of subunits during PHF assembly, could be absent or occluded by other phosphates present in the tau purified from cycled MT. Likewise, the increased phosphorylation of tau incorporated into SF and PHF could contribute to increased structural rigidity, analogous to the increase in the rigidity of tau paracrystals induced by phosphorylation (Hagestedt et al., 1989). We believe it is likely that if the phosphorylation state of SF and PHF could be duplicated, then identical structures would form under our assembly conditions.
Using the conditions for tau polymerization defined in our study has several potential advantages over previously reported procedures. First, reasonable filament yields were obtained at relatively low tau concentrations (1-10 µM). Previous studies have used concentrations of 50 µM (<10 filaments/field; Montejo de Garcini and Avila, 1987) to 250 µM (Crowther et al., 1994). One study did report assembly at a tau concentration of 2.5 µM, but only in the presence of transglutaminase (Dudek and Johnson, 1993). Since this enzyme could potentially induce polymer accumulation in a manner independent of physiologically relevant post-translational modifications, this protocol might not be suitable for defining those protein modifications which result in changes in filament morphology or rates of assembly. Second, unlike conditions reported for the assembly of tau fragments (Wille et al., 1992; Crowther et al., 1992), the conditions defined in this study are conducive to assembly of the full-length tau protein. This is important if the contribution of all regions of the tau sequence to the assembly process are to be assessed. Third, use of our assembly conditions results in the polymerization of a morphologically homogeneous population of filaments. Therefore, analyzing changes in morphology resulting from protein modification may be more straightforward than when using conditions which are reported to result in heterogeneous filament populations (Montejo de Garcini et al., 1986; Wille et al., 1992; Crowther et al., 1992, 1994). Finally, if chemical or enzymatic treatments of tau are to be screened for their potential ability to modulate tau polymerization in vivo, they should preferably be examined under conditions as close to physiological as possible.