anionic micelles and vesicles induce tau fibrillization in vitro

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Anionic Micelles and Vesicles Induce tau Fibrillization in vitro

Carmen N. Chirita‡§¶, Mihaela Necula‡§¶, and Jeff Kuret§R

From the ‡Biophysics Program; and §Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210

RTo whom correspondence should be addressed: Jeff Kuret, Ph.D. Center for Biotechnology 1060 Carmack Rd Columbus, OH 43210 TEL: (614) 688-5899 FAX: (614) 292-5379 Email: [email protected]

Running title: Micelle-Dependent tau Fibrillization

Keywords: tau, Alzheimer’s disease, tauopathies, polymerization inducers, free fatty acids,

arachidonic acid, phospholipids, phosphatidylserine, detergents

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 1, 2003 as Manuscript M301663200 by guest on A

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Abstract

Alzheimer’s disease is defined in part by the intraneuronal accumulation of filaments comprised of

the microtubule associated protein tau. In vitro, fibrillization of recombinant tau can be induced by

treatment with various agents, including phosphotransferases, polyanionic compounds, and fatty

acids. Here we characterize the structural features required for the fatty acid class of tau

fibrillization inducer using recombinant full-length tau protein, arachidonic acid, and a series of

straight chain anionic, cationic, and nonionic detergents. Induction of measurable tau fibrillization

required an alkyl chain length of at least 12 carbons and a negative charge consisting of carboxylate,

sulfonate, or sulfate moieties. All detergents and fatty acids were micellar at active concentrations,

owing to a profound, tau-dependent depression of their critical micelle concentrations. Anionic

surfaces larger than detergent micelles, such as those supplied by phosphatidylserine vesicles, also

induced tau fibrillization with resultant filaments originating from their surface. These data suggest

that anionic surfaces presented as micelles or vesicles can serve to nucleate tau fibrillization, that

this mechanism underlies the activity of fatty acid inducers, and that anionic membranes may serve

this function in vivo.

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Introduction

Alzheimer’s disease (AD)1 is a progressive neurodegenerative disease characterized in part by a

constellation of intracellular neurofibrillary lesions termed neurofibrillary tangles, neuritic plaques,

and neuropil threads (1). Each manifestation of neurofibrillary pathology is comprised of tau

protein polymerized into filaments. Because neurofibrillary lesions appear with a stereotypic spatial

distribution (2,3) and correlate with both neuronal cell loss (4) and cognitive decline (5), they are

useful markers of degeneration in AD and other dementias.

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), its state of postranslational modification (8,9), by the

addition of polyanionic substances such as sulfated glycosaminoglycans (heparin, dextran sulfate,

pentosan polysulfate; Refs. (10-12), 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) in aqueous solution, fatty acids form micelles

wherein 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


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motifs (17). Although such motifs have been suggested to exist in α-synuclein (18), a protein that

forms amyloid filaments in Parkinson’s disease (19-21), they have not been found in human tau

protein.

Here we examine the importance of alkyl chain length, chemical nature of the charged

headgroup, 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 Materials. Recombinant, His-tagged tau protein (htau40) was purified as described (22).

Arachidonic acid (AA) and 14C-labeled AA (55 Ci/mol) were obtained from Cayman Chemicals

(Ann Arbor, MI) and American Radiolabeled Chemicals (St Louis, MO), respectively, and stored at

-80°C under argon. Palmitoleic and stearic acids were from Sigma (St. Louis, MO). Alkyl sulfate

detergents (12 – 20 carbons) were obtained from Mallinckrodt (Paris, KY), Acros Organics (Morris

Plains, NJ), Lancaster Synthesis (Pelham, NH), and Research Plus (Bayonne, NJ) as sodium salts.

Alkyl sulfonate detergents (6 – 18 carbons) were obtained as sodium salts from Research Plus.

Non-ionic and cationic detergents (bromide salts) were purchased from Sigma (C10E8, C12E23,

C14E8, and C21H46BrN), and Fluka (Milwaukee, WI; C8E4, C10E6, C19H42BrN, and C15H34BrN).

Porcine brain L-α-phosphatidylserine (containing mostly 18:0 and 18:1 fatty acid chains) was from

Avanti Polar-Lipids (Alabaster, AL). All detergent stock solutions were prepared in water, DMSO,

or 1:1 water isopropanol and stored at -20°C. N-phenyl-1-naphthylamine was from Sigma.

Tau Polymerization Assays. Under standard conditions, htau40 (4 - 8 µM) was incubated with AA

or other polymerization inducers (1 – 500 µM) in Assembly Buffer (10 mM HEPES, pH 7.4, 100

mM NaCl, 5 mM DTT) at either room temperature or at 37°C for 3 – 6 h. Samples were processed

for electron microscopy, fluorescence spectroscopy, or ultracentrifugation as described below.

Electron Microscopy. Aliquots 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 (King et al., 1999). Images were

viewed in a Phillips CM 12 transmission electron microscope operated at 65 kV. Random images

were captured on film at 8,000 – 22,000x magnification, digitized at 600 dots-per-inch resolution,


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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.

Ultracentrifugation. Tau polymerization reactions were centrifuged (400,000g X 30 min) in an

Optima TLX ultracentrifuge (TLA 100.2 rotor). The resultant pellets were washed three times with

Assembly Buffer 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 Stoichiometry. Tau 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 assayed for protein and for 14C-AA by scintillation spectroscopy.

CMC Measurements. Detergents or lipids suspended in Assembly Buffer at varying concentrations

(1 µM – 10 mM) were incubated 1 h (37oC or room temperature) in the presence of 10 µM N-

phenyl-1-naphthylamine after which fluorescence intensity was read directly in a PTI fluorimeter

(λex = 346 nm; λem = 420 nm; 1 nm bandwidth, 800 V, gain 12.5, and slit 16) jacketed at 37 oC.

When present, htau40 was maintained at 4 µM. CMCs were estimated from abscissa intercepts

after least squares linear regression as described previously (24), and were 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 40ºC were calculated as described

previously (25).

Liposome and Mixed Micelle Preparations. Phosphatidylserine 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


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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.

Nomenclature. Polyoxyethylene 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 Anionic but not Nonionic or Cationic Detergents can Induce tau Protein Assembly. The

importance of alkyl chain length, headgroup charge, and CMC for induction of recombinant His-

tagged htau40 polymerization was examined for a selection of ionic and nonionic synthetic

detergents. His-tagged htau40 was used because of ease and yield of preparation and because the

presence of the tag does not significantly change the rate or extent of AA-induced tau fibrillization

(26). Nonionic detergents tested included the polyoxyethylenes Triton X-100 (data not shown),

Tween 20, C8E4, C10E6, C10E8, C12E23, and C14E6. These had CMC values ranging from 10 to 600

µM when measured under standard tau assembly conditions in the presence or absence of htau40

(Table I). None of the nonionic detergents were capable of inducing tau fibrillization (using the

transmission electron microscopy assay) when tested at concentrations up to 500 µM. In addition,

tau protein had no observable effect on detergent micellization (Table I). These results suggest that

uncharged surface-active hydrophobic agents were insufficient to promote tau fibrillization when

present in either dispersed or micellar form.

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 100 µM concentration (Fig. 1). To determine whether other

negatively charged chemical groups could substitute for the carboxylic acid moiety found AA, the

analysis was extended to include synthetic alkyl sulfate and alkyl sulfonate detergents containing


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12 – 20 saturated straight chain carbon atoms. Very few filaments of long length (��-4

filaments per 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, and therefore 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

��

induce tau fibrillization by similar mechanisms, and that the minimum structural features

responsible for measurable inducer activity are an alkyl chain at least 16 carbons in length and a

negatively charged head group comprised of at least carboxylate, sulfate, or sulfonate moieties.

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


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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 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) owing to the presence of neutral electrolyte (100 mM

NaCl) in the buffer (27). When measured in Assembly Buffer complete with htau40, however,

CMC values depressed still further, so that they were fully two orders of magnitude below values

observed in water alone (Fig. 4). Plots of log CMC vs. 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.

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. 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.


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Minimum Requirements for Micelle-mediated tau Fibrillization. To 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

its CMC under assembly conditions was low micromolar 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.

Phosphatidylserine Liposomes Induce tau Polymerization. To determine whether anionic lipids

could substitute for anionic detergents as inducers of tau fibrillization, htau40 was incubated (3 h at

37ºC) with 10 – 400 µM phosphatidylserine vesicles under standard conditions and subjected to

electron microscopy assay. Unlike anionic micelles, phosphatidylserine vesicles were readily

observable in electron microscopy assays owing to their large size (typically >50 nm diameter

compared to <10 nm diameter for detergents). Moreover, their CMCs in aqueous solution have

been estimated in the nanomolar range (29), so that they were almost completely vesicular prior to

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


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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.

AA-mediated tau Fibrillization Follows a Ligand-facilitated Mechanism. To test this hypothesis,

htau40 was subjected to fibrillization conditions for 3.5 h in the presence and absence of 14C-labeled

AA (75µM), 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 per mole 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 Here we examined the mechanism of fatty acid mediated tau fibrillization and showed that it

stems from two structural features: an alkyl chain of at least 12 carbons in length and a negatively

charged headgroup. Although requirement of an alkyl chain has been reported previously (14), its

role has been confounding because all fatty acids examined to date have been active well below

their CMCs, suggesting that alkyl chains were involved in binding tau protein rather than

micellization. Published CMC values were determined in the absence of tau protein, however, and

the surprising finding here is that the presence of even micromolar concentrations of tau protein

greatly depressed the CMC for AA and other anionic detergents. Protein-mediated depression of

alkyl sulfate CMC has been observed previously with micromolar concentrations of peptides

derived from uteroglobin (30), and result from electrostatic interactions between positively charged

amino acid sidechains and negatively charged fatty acids or detergents. Indeed, a single pendent

alkyl amine (grossly resembling a Lys residue when protonated) attached to a neutral analyte such

as polyethyleneglycol can depress the CMC for fatty acids by an order of magnitude (31). The

htau40 construct used here contained a total of 59 Lys and Arg residues and a predicted net charge

of +3.51 at assay pH (7.4). Because htau40 had no effect on the CMC of nonionic detergents, it

does not appear to depress CMC by modulating solvent surface tension (32). In any event, AA and

anionic detergents are mostly micellar at 100 µM in the presence of tau protein. Inducer activity

appears to reside with the micelle, because incubation of tau with preformed micelles or lipid

vesicles results in tau fibrillization. Moreover, tau filament yield initially increases with detergent

concentration above the CMC, which corresponds to an increase in micelle but not detergent

monomer concentration (28). Therefore it is concluded that the principal role of the alkyl chain is

to support micellization, and micelle formation in the 50 – 100 µM range in vitro requires an alkyl


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chain of at least 12 carbons. The alkyl moiety can be saturated (14) or unsaturated (herein and Ref.

(14), and can be part of fatty esters such as phospholipids (herein).

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 (10-13,33,34). 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

sedmentation.

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


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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 prote�� !�� !��

protein, apolipoprotein C-""" ��-synuclein can obliquely insert into lipid membranes in a viral

peptide fashion (40-44) and this may contribute to their lipid-dependent aggregation (45-51). 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 gives 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 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 is heavily complexed with protein and


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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 AD 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, since 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 - 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 while 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 so 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


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specimens of authentic AD 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-dependency (6,60) result from two simultaneous

reactions: tau promoting the cooperative micellization of initially dispersed AA, and AA micelles

promoting the fibrillization of tau. Moreover, the biphasic nature of the AA concentration

dependence (herein 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 suggests that anionic

membranes are candidate nucleation centers in vivo.


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Acknowledgments

We thank Dr. Robert Lee, OSU College of Pharmacy, for guidance in liposome preparation, and

Drs. T. Chris Gamblin and Aida Abraha, Northwestern University Medical School, for guidance

with electron microscopy methods.


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FOOTNOTES

¶ These authors contributed equally to this work.

*This work was supported by National Institute of Health grant AG14452 (to J.K.)

1The abbreviations used are: AD, Alzheimer's disease; AA, arachidonic acid; CMC, critical micelle

concentration; and Tween 20, Polyoxyethylene sorbitan monolaureate


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Figure Legends Fig. 1: Alkyl Sulfates Induce tau Fibrillization. Htau40 (4 µM) was incubated (3 h at 37oC) in the

presence of 100 µM A, AA; B, C20H41NaSO4; or C, C18H37NaSO4 and then examined by

transmission electron microscopy at 8,000-fold magnification. AA and both alkyl sulfate detergents

induced straight filaments, which differed in number and length with each inducer. Bar = 500 nm.

Fig. 2. Alkyl Sulfates Resemble AA in Potency. Htau40 (4µM) was incubated (3 h at 37oC) in the

presence of varying concentrations of AA (�# � $18H37SO4Na (• ), or C20H41SO4Na (o) and then

examined by transmission electron microscopy at 22,000-fold magnification. Filaments from two

negatives were measured, summed, and plotted as total filament length ± range.

Fig. 3. Length Distributions of AA and Alkyl-sulfate Induced tau Filaments. Htau40 (4 µM) was

incubated (3 h at 37oC) in the presence of 75 µM AA (�# �$18H37SO4Na (• ), and C20H41SO4Na (o)

and then examined by transmission electron microscopy at 8,000-fold magnification. The lengths

of all filaments ��%�� &��!��'��

point represents the percentage of all analyzed filaments (totaling 1580, 2067, and 591 for AA,

C18H37SO4Na, and C20H41SO4Na, respectively) that segregated into consecutive length intervals

(100 nm bins), whereas each line represents the best fit of the data points to an exponential

distribution. Although exponential distributions were observed for all inducers at 75 µM,

C18H37SO4Na, and C20H41SO4Na induced fewer filaments that grew to longer lengths relative to

AA.

Fig. 4. CMC Values for Alkyl Sulfate Detergents. CMC values for alkyl sulfate detergents were

determined (37oC) in Assembly Buffer in the presence (�#��(�) of htau40 (4 µM) and

compared to published reference values (25) calculated in water at 40ºC (�#��


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conditions confirmed an inverse log-linear relationship between alkyl chain length and CMC (solid

lines), the presence of tau greatly lowered measured CMC values relative to water or buffer alone.

Figure 5. Dependence of tau Fibrillization on Anionic Micelles. Tau fibrillization was induced

by incubation of htau40 (4 µM) with mixed micelles (100 µM detergent) prepared from non-ionic

detergent C14E8 and varying mol % C20H41SO4Na. The percentage of total tau mass recovered in

the pellet fraction after ultracentrifugation (30 min x 400,000g) was measured and expressed as an

average % recovery ± S.D. of replicate experiments. Data points between 40 – 100 mol %

C20H41SO4Na were fit by least squares linear regression (solid line). This line intersected the

abscissa at 38.0 ± 6.4 mol %, yielding an estimate of the minimum concentration of C20H41SO4Na

required to support tau fibrillization.

Figure 6. Stimulation of tau Fibrillization by Anionic Lipid. Htau40 (4 µM) was incubated (3 h at

37ºC) with preformed phosphatidylserine vesicles and then examined by transmission electron

microscopy (22,000-fold magnification). Vesicles were observed as bodies >50 nm in diameter

(arrows) that were frequently associated with filaments. Approximately 15% of well-resolved

filaments were found extending from the surface of vesicles (asterisk). Bar = 100 nm.


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Table I

Detergent CMC values measured at 37ºC

Detergent CMC - htau40 + htau40a

(µM) (µM)

Polyoxyethelene Nonionic Detergents

C8E4 586 ± 48 NDb

C10E6 609 ± 33 ND

C10E8 627 ± 120 ND

C12E23 49.5 ± 18.6 52.2 ± 12.8

Tween20 24.7 ± 2.3 26.0 ± 3.1

C14E8 11.3 ± 3.4 8.9 ± 1.8

Quaternary ammonium cationic detergents

C15H34BrN 7,600 ± 100 ND

C19H42BrN 88.1 ± 3.4 38.9 ± 7.1

C21H46BrN 18.6 ± 4.5 19.5 ± 0.9

Fatty acids

palmitoleic acid 996 ± 26 87.3 ± 3.6

stearic acid 393 ± 2 50.0 ± 17.1

Arachidonic acid 236 ± 12 8.1 ± 0.5

aMeasured in presence of 4 µM htau40.

bNot determined.


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Table II

Association of 14C-AA with tau Filaments After Centrifugation

Sample aProtein Protein Tau bAA Stoichiometry

(��# (%) (nmol) (nmol) (mol AA/mol tau)

total 218 ± 24 100 ± 11 4.54 ± 0.48 ---- ----

pellet 106 ± 6 49 ± 3 2.21 ± 0.24 0.41 ± 0.11 0.19 ± 0.05

supernatant 115 ± 12 53 ± 6 2.40 ± 0.26 22.26 ± 0.34 9.28 ± 1.10

aDetermined by Coomassie Blue binding assay as described in Experimental Procedures. Data are

reported as average ± S.D. of three independent experiments measured in duplicate.

bDetermined using 14C-AA as described in Experimental Procedures. Data are reported as average

± S.D. of two independent experiments measured in duplicate.


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Carmen N. Chirita, Mihaela Necula and Jeff KuretAnionic micelles and vesicles induce tau fibrillization in vitro

published online May 1, 2003J. Biol. Chem.

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