From the
Biophysics Program and
Department of Molecular and Cellular
Biochemistry, The Ohio State University College of Medicine and Public Health,
Columbus, Ohio 43210
Received for publication, February 17, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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There is much interest, therefore, in identifying cellular components that initiate tau filament formation in disease. Fibrillization of recombinant, full-length tau protein in vitro does not occur spontaneously at physiological concentrations (6). However, tau protein can be induced to fibrillize by changes in its primary structure (7) or its state of posttranslational modification (8, 9); by the addition of polyanionic substances such as sulfated glycosaminoglycans (heparin, dextran sulfate, and pentosan polysulfate) (1012), polyglutamate (11), and RNA (13); or by addition of fatty acids (14). Of these, fatty acids are especially efficacious in promoting the fibrillization of full-length tau protein at near physiological pH, temperature, reducing environment, ionic strength, and tau protein concentration (14, 15).
Nonetheless, the mechanism by which fatty acids induce tau fibrillization
is unkown. Fatty acids resemble detergents in having hydrophobic alkyl chains
and charged (anionic) head groups. Above their critical micelle concentrations
(CMCs)1 in aqueous
solution, fatty acids form micelles in which their hydrophobic moieties are
sequestered, and their charged head groups are exposed to solvent. We
(6) and others
(16) have argued that the
behavior of fatty acids such as arachidonic acid in assays of tau aggregation
was consistent with it acting in micellar form. Yet fatty acids induce tau
fibrillization at concentrations well below their measured CMC values
(14). As free monomers, fatty
acids have been shown to reversibly bind proteins through specific
high-affinity motifs (17).
Although such motifs have been suggested to exist in -synuclein
(18), a protein that forms
amyloid filaments in Parkinson's disease
(1921),
they have not been found in human tau protein.
Here we examine the importance of alkyl chain length, chemical nature of the charged head group, and CMC for induction of tau polymerization using arachidonic acid, a series of ionic and nonionic synthetic detergents, and the anionic lipid phosphatidylserine. The results show that fatty acids induce tau fibrillization in micellar form without stoichiometric incorporation into filaments. The micelles must be negatively charged to promote fibrillization of full-length tau protein and, in the case of alkyl sulfate detergents, must contain at least 38 mol % negatively charged species. Because anionic lipids also induce tau fibrillization, it is proposed that intracellular membranes represent a class of physiologically relevant, intracellular tau polymerization inducers.
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EXPERIMENTAL PROCEDURES |
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Tau Polymerization AssaysUnder standard conditions, htau40 (48 µM) was incubated with AA or other polymerization inducers (1500 µM) in Assembly Buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, and 5 mM dithiothreitol) at either room temperature or 37 °C for 36 h. Samples were processed for electron microscopy, fluorescence spectroscopy, or ultracentrifugation as described below.
Electron MicroscopyAliquots of tau polymerization reactions were taken, treated with 2% glutaraldehyde (final concentration), mounted on formvar/carbon-coated 300 mesh grids, and negatively stained with 2% uranyl acetate as described previously (6). Images were viewed in a Phillips CM 12 transmission electron microscope operated at 65 kV. Random images were captured on film at x8,000 to x22,000 magnification, digitized at 600 dots-per-inch resolution, and imported into Optimas 6.5.1 for quantification of filament lengths and numbers (6). Individual filaments were defined as any object greater than 50 nm in its long axis and were counted manually.
UltracentrifugationTau polymerization reactions were centrifuged (400,000 x g, 30 min) in an Optima TLX ultracentrifuge (TLA 100.2 rotor). The resultant pellets were washed three times with Assembly Buffer and then dissolved in 5% C12H25SO4Na containing 0.01 M NaOH. The amount of protein in the supernatant and pellet fractions was determined by the Coomassie Blue binding method (23) using purified, recombinant htau40 as standard.
Arachidonic Acid Binding StoichiometryTau protein (8 µM) was subjected to polymerization as described above, except that AA was 14C-radiolabeled at a final specific activity of 25 Ci/mol. Reaction products were subjected to ultracentrifugation, and aliquots of the resultant supernatants and resuspended pellets were assayed for protein and [14C]AA by scintillation spectroscopy.
CMC MeasurementsDetergents or lipids suspended in Assembly
Buffer at varying concentrations (1 µM to 10 mM) were
incubated for 1 h (at 37 °C or room temperature) in the presence of 10
µM N-phenyl-1-naphthylamine, after which fluorescence
intensity was read directly in a PTI fluorimeter (ex = 346
nm,
em = 420 nm, 1 nm bandwidth, 800 V, gain 12.5, and slit
16) jacketed at 37 °C. When present, htau40 was maintained at 4
µM. CMCs were estimated from abscissa intercepts after
least squares linear regression as described previously
(24) and expressed as CMC
± S.E. of the estimate. Only data points within 5-fold of the CMC were
used to calculate CMC.
CMC values for alkyl sulfate detergents in water at 40 °C were calculated as described previously (25).
Liposome and Mixed Micelle PreparationsPhosphatidylserine was dissolved in chloroform at 1 mM, dried under a stream of argon (10 min), and dried by vacuum desiccation (45 min). Dried samples were hydrated at 1 mM concentration in Assembly Buffer by bath sonication in a Parafilm sealed glass test tube for 3 h. Liposomes were prepared fresh for each experiment, and their presence was confirmed by electron microscopy.
Mixed detergent micelles were prepared by mixing varying ratios of C14E8 and C20H41SO4Na at 2 mM total concentration followed by 30 min of sonication. Fibrillization reactions were performed as described above with 100 µM total final detergent concentration.
NomenclaturePolyoxyethylene detergents of formula CH3(CH2)y-O(CH2CH2) x-H are referred to as C(y+1)Ex, where y and x are the number of methylene and oxyethylene groups, respectively. Sodium alkyl sulfate and alkyl tetramethyl ammonium bromide detergents are referred to by their chemical formulas.
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RESULTS |
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Extending the analysis to cationic detergents C15H34BrN, C19H42BrN, and C21H46BrN revealed that they too were inactive as fibrillization inducers when assayed above or below their CMC, although the presence of tau protein modestly depressed the CMC for C19H42BrN (Table I). These data suggest that, like nonionic detergents, positively charged ionic detergents are incapable of inducing tau fibrillization in either dispersed or micellar form.
In contrast to these results, AA, which shares chemical properties with
anionic detergents, was a powerful inducer of tau fibrillization at 75
µM concentration (Fig.
1). To determine whether other negatively charged chemical groups
could substitute for the carboxylic acid moiety found in AA, the analysis was
extended to include synthetic alkyl sulfate and alkyl sulfonate detergents
containing 1220 saturated straight chain carbon atoms. Very few
filaments of long length (≥500 nm; 14 filaments/grid) were observed
in the electron microscope when using the 12- or the 14-carbon sulfate or
sulfonate series detergents as inducers. In contrast, alkyl sulfate and
sulfonate detergents containing 16, 18, and 20 carbons induced significant
polymerization. C18H37SO4Na and
C20H41SO4Na were the most active inducers
among this series; therefore, they were used in the studies described below.
They produced abundant straight filaments from recombinant htau40 that were
morphologically similar to those induced by AA
(Fig. 1). Moreover, the alkyl
sulfate detergents were broadly similar to AA in potency, yielding biphasic
dose-response curves with maximal filament mass yielded at concentrations
between 50 and 150 µM (Fig.
2). Nonetheless, alkyl sulfate inducers differed quantitatively
from AA in that they appeared to be weaker nucleating agents, producing a
smaller number of filaments that achieved longer length
(Fig. 1). This was apparent in
filament length distributions, which remained exponential but skewed toward
longer lengths when induced by alkyl sulfates
(Fig. 3). For example, AA
produced >10-fold more filaments than
C20H41SO4Na that were on average >5-fold
shorter in length. Overall, despite inducing far fewer filaments, the total
mass of filaments formed from C20H41SO4Na was
typically 75% of the mass induced by AA. These data suggest that AA and
the alkyl sulfate detergents induce tau fibrillization by similar mechanisms
and that the minimum structural features responsible for measurable inducer
activity are an alkyl chain of at least 16 carbons in length and a negatively
charged head group comprised of at least carboxylate, sulfate, or sulfonate
moieties.
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AA-induced Tau Fibrillization Requires Micelle Formation CMC values for AA and alkyl sulfate detergents were measured to determine whether micelles were important for tau fibrillization activity. Consistent with earlier observations (14), the CMC for AA in Assembly Buffer alone was measured as 236 ± 12 µM, which was well above the concentration required for tau fibrillization. When CMC was measured in Assembly Buffer complete with htau40 at 4 µM, however, the CMC decreased to 8.1 ± 0.5 µM. Tau-mediated CMC depression was observed with other fatty acids as well (palmitoleic and stearic acids; Table I), indicating that the effect was not unique to AA and extended to both saturated and unsaturated fatty acids. These data show that fatty acids aggregate to form micelles at a much lower concentration in the presence of htau40 than in its absence. CMC depression was apparent even at substoichiometric molar ratios of htau40 to fatty acid.
These observations were extended to a series of alkyl sulfate detergents, which follow a log-linear relationship between CMC and alkyl chain length when analyzed in water (Fig. 4; Ref. 25). The slope of this relationship is proportional to the free energy contribution of transferring methylene groups from solvent to micelles (25). When measured in the presence of Assembly Buffer (without tau), the relationship between log CMC and alkyl chain length remained linear but was depressed toward lower CMCs (Fig. 4) because of the presence of neutral electrolyte (100 mM NaCl) in the buffer (27). When measured in Assembly Buffer complete with htau40, however, CMC values were depressed still further, so that they were fully 2 orders of magnitude below the values observed in water alone (Fig. 4). Plots of log CMC versus alkyl chain length remained linear under these conditions, with slopes that were statistically indistinguishable (95% confidence interval) from those obtained in Assembly Buffer alone (Fig. 4), suggesting that the detergent aggregates formed in the presence of tau protein resembled authentic micelles with respect to their free energies of formation.
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These data show that tau protein exerted a profound effect on the micellization of anionic detergents and that anionic micelles were present under all assembly conditions that yielded tau filaments. The observation that tau fibrillization was initially proportional to increasing detergent or fatty acid concentration above their true CMCs (Fig. 2), where micelle but not monomer concentration was increasing (28), suggests that tau fibrillization is proportional to the concentration of anionic detergent or fatty acid micelles.
Minimum Requirements for Micelle-mediated Tau FibrillizationTo quantify the importance of negative charge for tau fibrillization, htau40 was incubated with mixed micelles prepared from nonionic detergent C14E8 and varying mol % concentrations of ionic inducer C20H41SO4Na. C14E8 was chosen as carrier because it was incapable of inducing tau fibrillization on its own and because, under assembly conditions, its had a low micromolar CMC, regardless of whether htau40 was present (Table I). Total detergent concentration was held constant at 100 µM to ensure the presence of micelles under all assay conditions, and resultant total filament mass was estimated by assaying tau protein in pellet and supernatant fractions after ultracentrifugation. Tau aggregation was not detectable below 20 mol % C20H41SO4Na but was observed at 40 mol % and increased above that concentration to 100 mol % C20H41SO4Na (Fig. 5). At that point, 39% of total tau protomer was rendered insoluble. Assuming a linear relationship between C20H41SO4Na content and tau filament formation and extrapolating to the ordinate intercept, tau fibrillization was supported under standard conditions when 100 µM mixed micelles contained 38.0 ± 6.4 mol % C20H41SO4Na.
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Phosphatidylserine Liposomes Induce Tau PolymerizationTo determine whether anionic lipids could substitute for anionic detergents as inducers of tau fibrillization, htau40 was incubated (3 h at 37 °C) with 10400 µM phosphatidylserine vesicles under standard conditions and subjected to electron microscopy assay. Unlike anionic micelles, phosphatidylserine vesicles were readily observable in electron microscopy assays because of their large size (typically >50 nm in diameter compared with <10 nm in diameter for detergents). Moreover, their CMCs in aqueous solution have been estimated in the nanomolar range (29), so that they were almost completely vesicular before incubation with tau protein. Examination of reaction products by electron microscopy showed the presence of vesicles and very long filaments at most phosphatidylserine concentrations tested. Closer inspection revealed that at least 15% of all filaments were associated with phospholipid vesicles through their ends, which appeared to extend from the vesicle surface (Fig. 6). Other vesicles were observed alone or associated with filaments along their length (Fig. 6). These data showed that anionic vesicle-forming lipids were capable of inducing tau filament formation and suggested that the mechanism involved facilitation of tau aggregation at the vesicle surface.
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AA-mediated Tau Fibrillization Follows a Ligand-facilitated
MechanismTo test this hypothesis, htau40 was subjected to
fibrillization conditions for 3.5 h in the presence and absence of
[14C]AA (75 µM), and the amount of labeled AA
comigrating with filamentous tau was determined after centrifugation. Under
these conditions, 50% of AA-treated htau40 comigrated with the pellet
(filamentous) fraction, whereas most AA (>97%) remained in the soluble
fraction (Table II). The pellet
fraction contained 0.19 ± 0.05 mol AA/mol tau, confirming that AA
remained at least partially associated with tau filaments but was not
incorporated with 1:1 molar stoichiometry with respect to tau protomer. These
data suggest that AA acted in micellar form to facilitate tau aggregation but
did not directly mediate filament extension with concomitant incorporation
into growing filaments.
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DISCUSSION |
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Inducer activity also requires an ionizable head group, with sulfate, sulfonate, and carboxylate moieties all supporting tau fibrillization, even when presented as part of a phospholipid head group, as in the case of phosphatidylserine. The resultant negative charges appear to supply more than simple amphiphilic character to promote micelle formation because cationic detergents of similar alkyl chain length and CMC do not support tau fibrillization. Micelle-forming nonionic detergents and uncharged methyl or ethyl esters of AA (14) also are inactive. Therefore, it appears that the key role of ionizable groups is to present a negatively charged surface on the micelle.
Anionic micelles may induce filament formation by concentrating the basic
tau protein molecules close to their surface, such that the energy barrier for
nucleation is overcome (6,
14). Similar mechanisms have
been postulated for the polyanion class of inducers, including heparin,
poly-glutamate, nucleic acids, and the microtubule surface
(1013,
33,
34). Alternatively, or in
addition, filament formation could stem from micelle-dependent stabilization
of assembly-competent protein conformations
(35). Indeed, protein
conformation alterations have been suggested to underlie observed differences
in the ability of protein kinases to phosphorylate tau in the absence or
presence of phospholipid liposomes
(36). Regardless of its
effects, micelle-tau association appears to be reversible because only
15% of mature filaments were found associated with liposomes and because
of the poor recovery of [14C]AA when tau filaments were isolated by
sedimentation.
Although all alkyl sulfate detergents examined form micelles above their
CMCs, their efficacy in promoting tau fibrillization under conditions reported
here varied widely. For example, 12- and 14-carbon alkyl sulfates are
extremely weak inducers and yield insufficient filaments to quantify by
current assay methods. Significant quantities of filaments can be observed by
electron microscopy using C16H33SO4Na, with
C18H37NaSO4 and
C20H41NaSO4 inducing large amounts of
filaments. Because the degree of ionization of alkyl sulfate micelles is
independent of aggregation number and salt concentration
(37), it is unlikely that
micelle charge varies with increasing alkyl chain length to influence anionic
detergent efficacy. In contrast, micelle aggregation number does increase
exponentially with alkyl chain length
(38) and can lead to major
differences in micelle size and shape. Extrapolating this relationship to
C16H33SO4Na predicts an aggregation number of
250 molecules/micelle near the CMC in solutions containing 100
mM NaCl (38). This
corresponds to a radius less than half the hydrodynamic radius of monomeric
tau protein (39). Efficient
induction of tau fibrillization above this size may be related to micelle
curvature, surface area, or volume. Although not demonstrated for tau protein,
other amyloid-forming proteins such as A
, prion protein, apolipoprotein
C-III, and
-synuclein can obliquely insert into lipid membranes in a
viral peptide fashion
(4044),
and this may contribute to their lipid-dependent aggregation
(4551).
Thus, the size of the hydrophobic micelle core may play a role in favoring
polymerization-prone conformations of partially inserted proteins. Finally, it
is noted that the large differences in aggregation number among alkyl sulfate
detergents give rise to vastly different relationships between micelle and
total detergent concentrations. This consideration may underlie the higher
potency of C18H37NaSO4 relative to
C20H41NaSO4
(Fig. 2), even though its CMC
is greater. It may also limit the apparent efficacy of the smaller alkyl
sulfate detergents by greatly narrowing the concentration range over which
they are active (see the discussion of biphasic behavior below). Further
experimentation will be necessary to determine which of these considerations
is most important for differences in alkyl sulfate efficacy.
Although polyanions have emerged as useful tools in vitro, their structures have not pointed toward a clear cellular agent that would serve to promote tau fibrillization in disease. For example, RNA is present at high concentration inside of cells, but it is heavily complexed with protein and presumably not available in free form in sufficient amounts to induce tau filament formation (52). Similarly, heparan sulfate proteoglycans are extracellular and sequestered from the bulk of physiological tau protein. In contrast, the activity of anionic micelles and vesicles points toward cellular membranes as naturally abundant intracellular sources of clustered negative charge. Studies with Alzheimer's disease tissue show that hyperphosphorylated tau colocalizes with lipid rafts (53) and that tau filaments appear in association with cytomembranes (54). In fact, membrane association may be a normal function of tau because it has been shown to occur upon heterologous expression in PC12 cells (55) and may be mediated by electrostatic interaction with anionic lipids. Phosphatidylserine, the most abundant anionic phospholipid, comprises between 10 and 20 mol % of total phospholipid in cell membranes (56). Because phosphatidylserine is distributed primarily on the cytoplasmic face of cellular membranes (57), substantial negative charge is potentially available for binding tau protein. Using purified components, we found that fibrillization of htau40 required at least 38 mol % anionic charge in vitro. These results suggest that whereas phospholipid membranes are potential sites of tau aggregation in vivo, normal levels of anionic phospholipids may not be sufficient to drive fibrillization. Rather, increased levels of anionic lipids (58, 59), increased free tau concentration, or fibrillization-promoting changes such as hyperphosphorylation may be required for them to serve this pathological function. We note that individual tau isoforms differ in their aggregation properties (26), and hence the minimum charge content required for assembly of the mixtures of tau isoforms found in vivo will likely differ from the value determined here for htau40 alone.
Fatty acids have been especially useful agents for studying tau polymerization because of their efficacy with full-length tau protein under near-physiological conditions (6, 26). Moreover, as shown here, tau filaments nucleated with anionic vesicles (and presumably anionic micelles) morphologically resemble tau filaments extending from cellular membranes observed in biopsy specimens of authentic Alzheimer's disease brain (compare Fig. 6 herein with Fig. 2 of Ref. 54), suggesting that lipid-induced tau fibrillization models an authentic pathological process. Yet the method has disadvantages. First is the susceptibility of unsaturated fatty acids such as AA to oxidation, which modulates their activity and eventually renders them inert (60). Although alkyl sulfates induce fewer filaments than AA, they are more water-soluble, stable, and cheaper and thus provide convenient alternatives for in vitro polymerization reactions. A second disadvantage has been the unusual kinetics associated with AA-induced tau fibrillization. It has been postulated that this results from association of preformed micelles with tau (16). But from the work presented herein, it is now clear that the rapid initial time-course of reaction (which has been modeled as a two-phase exponential reaction; Ref. 6) and cooperative AA dependence (6, 60) result from two simultaneous reactions: (a) tau promoting the cooperative micellization of initially dispersed AA, and (b) AA micelles promoting the fibrillization of tau. Moreover, the biphasic nature of the AA concentration dependence (this work and Ref. 6) likely results from increasing concentrations of micelles binding increasing proportions of total tau monomer, thereby inhibiting filament extension and, eventually, nucleation. Replacement of fatty acids with alkyl sulfates will not change these latter characteristics.
In summary, we have shown that arachidonic acid promotes tau fibrillization in micellar form and can be replaced by anionic detergents or phospholipids. The data suggest that anionic membranes are candidate nucleation centers in vivo.
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FOOTNOTES |
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¶ Both authors contributed equally to this work.
|| To whom correspondence should be addressed: Center for Biotechnology, 1060 Carmack Rd., Columbus, OH 43210. Tel.: 614-688-5899; Fax: 614-292-5379; E-mail: kuret.3{at}osu.edu.
1 The abbreviations used are: CMC, critical micelle concentration; AA,
arachidonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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