The organization of the microtubule associated protein tau in Alzheimer paired helical filaments

Brain Research, 602 (1993) 1-13

1

© 1993 Elsevier Science Publishers B.V, All rights reserved 0006-8993/93/$06.00

BRES 18332

Research Reports

The organization of the microtubule associated protein tau in Alzheimer paired helical filaments G e o r g e C. R u b e n

a

K h a l i d I q b a l b, I n g e G r u n d k e - I q b a l

b a n d J o h n E . J o h n s o n Jr. c

a Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 (USA), b New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY10314 (USA) and c Department of lntegrative Biology, University of Califonia, Berkeley, CA 94720 (USA) and Department of Neuroscience, SRl lnternational, Menlo Park, CA 94025 (USA) (Accepted 14 July 1992)

Key words: Alzheimer disease; D e n a t u r e d tau polymer filament; Freeze-dried Pt-C replicated neurofibrillay tangle; Neurofibrillary pathology

The structural relationship of the microtubule associated protein tau to paired helical filaments (PHF) was examined by high resolution transmission electron microscopy (TEM) without treatment with any chemical fixatives. Neurofibrillary tangles (NFT) were isolated in the absence of detergent from Alzheimer diseased brains, were freeze-dried, and were vertically platinum-carbon replicated for TEM. The P H F we observed made one helical turn ( L ) in 74 + 8.5 n m and had a wide region ( W ) of 14.8 +_0.6 n m similar to P H F previously modeled with a periodic morphology. The P H F thin region (T) m e a s u r e d ~ 2.4 nm, ~ 4.9 nm, ~ 7.4 nm and - 9.7 n m and the most often observed width was ~ 2.4 nm. No surface features regularly divide the P H F into two filaments. Morphologically the P H F are thin helical ribbons with an often observed thickness of ~ 2.4 nm. At high magnification, ~ 1.0 n m and some ~ 0.4 nm strands identical to normal and denatured tau m o n o m e r covered P H F surfaces and were aggregated in non-periodic fashion. Bovine tau polymer ~ 2.1 nm diameter filaments, trapped on a filter, were partially heat denatured and showed some of the morphological features of PHF.

INTRODUCTION Neurofibrillary tangles (NFT) of paired helical filaments are one of the most characteristic brain lesions of Alzheimer disease 2°,41 and their abundance determines the diagnosis. This histopathological hallmark occurs with greater frequency in patients with clinical dementia of the Alzheimer type than in normal individuals of the same age 2'42'46. Therefore, it is very important that we establish and understand the arrangement of subunits in paired helical filaments. This knowledge, we believe, could be key to understanding the pathogenesis of neurofibrillary degeneration. Intracellular Alzheimer neurofibrillay tangles fixed in osmium tetroxide or potassium permanganate in the absence of glutaraldehyde treatment were first observed by transmission electron microscopy (TEM) of thin sections and reported by Kidd 19'2°. The tangles he observed contained helical pairs of ~ 10 nm filaments separated center to center by 15 nm with a double helical period of ~ 160 nm that he named paired helical filaments (PHF). Subsequent thin sectioning

work with a similar fixation treatment confirmed that the P H F were helical with cross-over width, T-- 12.8 _+ 2 nm, and a wide region, W = 2 4 + 3 . 1 nm, which occurred every L -- 65-80 nm 53. Negatively stained isolated P H F were observed with cross-over widths of T = 6 - 1 1 nm, and wide regions of W = 15-22 nm which occured every L = 60-90 nm 5'47'52. In contrast to the thin sectioned P H F images, the wide regions in a normal negatively stained P H F image rarely split into two filaments. Periodic P H F structural models have been advanced within the constraints of the negative stain dimensions but none has garnered consensus status5,21,45, 48.

Tau has been found in abnormally phosphorylated forms in P H F and has been revealed as the integral protein subunit of P H F by both immunocytochemical labeling as well as protein compositional analysis of isolated N E T 10-12'16-18'24. Tau has also been shown to form ~ 2.1 nm triple-stranded left-hand helical polymers of ~ 1.0 nm strands which we think may be its native form 35. In addition, ~ 2.1 nm triple-stranded left-hand helical filaments structurally identical to tau

Correspondence: G.C. Ruben, D e p a r t m e n t of Biological Sciences, D a r t m o u t h College, Hanover, N H 03755, USA. Fax: (1) (603) 646-1347.

polymer filaments whave been found condensed in tangles with P H F 36. Although direct visualization of tau in P H F has been an important research objective for years, conventional sample preparation methods using thin sectioning and negative staining techniques for T E M have not achieved this goal 36. A new freeze-drying vertical replication method for T E M allows observation of single P H F and their upper surface within isolated tangles at a 0.6-0.7 nm resolution and a 260 nm depth of field. Resolution in replicas is directly related to the platinum-carbon (Pt-C) deposition angle to a surface and to the final Pt-C film thickness 34. Vertical replication not only produces high resolution replicas but this deposition method coats all flat surfaces with the same thickness while sloping surfaces receive a thinner Pt-C coat. Also, the steeper the slope, the thinner the Pt-C film TM. The ability to image P H F within isolated tangles provides the opportunity to determine with a new method whether normal P H F morphology consists of a pair of helical filaments with a periodic subunit structure, and to describe the handedness of the helical P H F in situ. By employing high resolution T E M of freeze-dried and vertically replicated NFT, we report the presence of 1.0 +_ 0.2 nm strands in P H F identical to tau m o n o m e r strands and we present evidence that ~ 2.1 nm tau polymer filaments can be transformed by partial heat denaturation into filaments with several PHF-like features. MATERIALS AND METHODS NFT preparations were isolated employing digestion with collagenase/dispase and in the absence of detergent as previously described 12'36. Bovine tau was isolated from twice cycled in vitro assembled brain microtubules as previously described 1°. Isolated bovine tau protein (50 p.g/ml, 150 mM NaCI) was trapped on a 0.1 /.tm Millipore filter and washed at 18-20°C with distilled water. One set of these samples was frozen, freeze-dried, and vertically replicated with 1.04 nm Pt-C and backed with 13 nm of evaporated carbon 35. In some cases, the tau samples were heated to 91°C on a water bath for 15 min, blotted and then frozen. This sample was similarly prepared as above with a 1.02 nm Pt-C film and a 13 nm carbon film thickness. Filament and polymer chain enlargement in vertically deposited Pt-C films has been empirically found by subtracting 4 A from the Pt-C film thickness measured on a quartz crystal monitor 34'36'37. Distances or spacings within a filament or a polymer chain are unchanged by vertical Pt-C replication. The transmission electron microscopy and image reversals have been previously described 34. The statistical treatment of the measurements in Table I used the normal distribution estimate of the means and the standard deviations were reported as the error in the mean. This same treatment was used to estimate the average of Table I values in Table II. The standard deviation was also reported as the error in the average of the means in Table II.

RESULTS The images of freeze-dried vertically replicated right-hand helical P H F within isolated N F T (Fig. 1)

look similar in size and shape to the P H F visualized by negative staining T E M 4752. Both vertically replicated and negatively stained P H F have the same helical turn period as thin sectioned P H F but the latter has greater width dimensions due to positive staining (Tables I and II). We have found four P H F thin region widths, T = 2.4 nm, 4.9 nm, 7.4 nm and 9.7 nm, but the P H F contain no regular protofilament or globular subunit structure corresponding to any of these sizes (Figs. 1-5). The substructure of the P H F is quite variable ranging from filaments with continuous surfaces (Figs. 2-4) to filaments with frequent holes in their surfaces (Figs. 1 and 3) 36, to filaments with variable numbers of protofilament-like lines in their surface, with a contour length shorter than 160 nm (Fig. 1). These observations suggest that the P H F are only partially regular at best. Second, none of the P H F surfaces show evidence of a double helical construction. The P H F were frequently adjacent to ~ 2.1 nm tau-polymer-like filaments (2.8 nm under a 1.14 nm Pt-C film) with left-hand helical ~ 1.0 nm strands (1.7 nm under a 1.14 nm Pt-C film) (Figs. 2c, 3a,b and 4a,b). This feature provided a very important resolution-size standard for judging P H F substructure. High magnification images of the P H F surfaces indicate that its substructure is non-periodically arranged and its surface is covered with 1.0 _+ 0.2 nm and 0.4_+0.2 nm strands (Figs. 3b and 4b and drawings in Figs. 3d and 4d). The P H F frequently contain thin regions that are similar in diameter to the ~ 2.1 nm diameter bovine tau polymer filaments (Fig. 5c) the surfaces of which contain 1.0 nm tau monomer strands 35. These observations suggested that the P H F could be a denaturation product of the ~ 2.1 nm triple-stranded left-hand helical tau polymer filaments which have been found in tangles 3~. This hypothesis was tested by using isolated bovine tau polymer immobilized on a 0.1 p,m Millipore filter similar to the tau polymer shown in Fig. 5c and characterized as highly purified tau 36. These tau polymer filaments of Fig. 5c were heated to 91°C on a water bath for 15 rain, blotted, frozen and replicated (1.02 nm thick Pt-C film) after freeze-drying. The resuits of this experiment are shown in Fig. 5a,b. The filaments in these images suggest that the denaturation of single ~ 2.1 nm tau polymer filaments can expand their width into the normally observed size range of the PHF. Only two regions in Fig. 5b appear to have roughly the same shape as the normal P H F wide regions (see denat, tau in Table I). Because these filaments were only partially denatured they contain thin regions (Fig. 5b) that average ~ 2.2 nm, - 4 . 4 nm, ~ 8.4 nm and ~ 10.4 nm as well as wider regions of 15.4 nm, 16.6 nm, 18.4 nm, 20.4 nm and 28.4 nm. These

partially denatured tau polymers have disordered surfaces containing 1.0 nm strands and some 0.4 + 0.2 nm strands similar to the P H F (figure not shown).

Comparison of PHF dimensions measured by vertical replication, negative staining and thin sectioning TEM Measurements of P H F from Figs. 1-4 and from previous reports are compared in Table I. The measurements of freeze-dried and vertically Pt-C replicated P H F in N F T were corrected for their Pt-C coati n g 34'36'37. The maximum error in any of these measurements would be 0.6-0.7 nm or equal to the correction for the Pt-C coating, however, we think the error is much less ( ~ 0.1-0.2 nm) 37. These PHF, unlike those isolated for negative staining, have remained in the isolated whole NFT and have not undergone sonication a n d / o r detergent treatment, which are often used

in P H F isolation and solubilization 5't7'47'52. The thin sectioned Alzheimer brain tissue samples reported in Table I were generally prepared and positively stained as outlined in Ruben et al. 36. The negative stain measurements recorded in Table I were measured directly from the published micrographs, in contrast to the thin sectioned P H F measurements where the authors' reported values are recorded. Measurements from the three preparation techniques are summarized in Table II. The average helical turn, L = 74.5 nm, varies by no more than 2% for all three methods, reinforcing our belief that P H F filaments were indeed measured in all cases. The average width of the thin regions and the wide regions are very similar in the vertically replicated and the negatively stained PHF, but clearly differ from their average thin section values (Table II). Thin sec-

Fig. 1. A NFT of PHF. The PHF labeled with large arrows can be followed for a distance of 0.2-0.6/~m. The distance between thin regions has been marked along these filaments and it averages L = 75.2+ 14.3 nm (n = 11) with PHF thick and thin widths of W = 14.6_+2.1 nm (n = 11) (15.2_+2.1 nm with a 0.6 nm Pt-C coating) and T = 2.7_+0.5 nm (n = 13) (3.3_+0.5 nm with a 0.6 nm Pt-C coating), respectively. This tangle also contains one ~ 2.1 nm tau-polymer-like filament ( ~ 2.8 nm with 0.6 nm of Pt-C added) marked with 2.8 nm as well as a region of amorphous material, marked A. The PHF in this NFT surface are right-hand helical or of indeterminant helicity. The PHF thickness is ~ 2.7 nm, the same as the cross-over region width, and these PHF frequently have a thin porous appearance that has been seen before (see Fig. 3c) 36. Also these PHF often split into short regions of protofilaments that never extend farther than one or two helical turns (see filament below 60 nm lettering and filament above the letter A at lower left). The PHF protofilament substructure in the figure occurs infrequently and it rarely appears to be regular. At the upper right center is the rare circumstance of two PHF (labelled 1 and 2) which twist around each other for only 200 nm.

TABLE I

Dimensions of certically replicated, negaticely stained and thin sectioned paired helical filaments

T T (nm)

W (nm)

2.7 ± 0.5 2.6±0.3 2.5 + 0.4 7.1+-1.4 9.8+-0.2 7.7±0.8 3.2 3.6 2.6±0.8

(n = 13) (n = 3) (n = 2) (n=5) (n = 3) (n = 2)

(n = 4)

9.6+ 1.4 2.1 +-0.4 4.9+-0.6 2.4_+0.2 7.9+-0.3

(n (n (n (n (n

4.5 ± 0.5 3.6+__0.5 5.4+ 1 5.3-+0.6 4.4±0.4 6-9 10 8-10 12.8±2

= = = = =

8) 4) 4) 5) 6)

14.6 +_2.1 15.1 +- 1.2 14.3 15.1.+1.2 14.5±1.2 15.1 -+0.9 16 17 16.0 ± 0.9

L (nm) (n = 11) (n = 2) (n=6) (n = 2) (n = 2)

(n = 9)

75.2 ± 14.3 70 + 1 82 59 _+ 8 78 ± 3 64 + 7 45 56 83.8± 15.7

(n = 11) (n = 2) (n=4) (n = 2) (n = 2)

(n = 10)

Source *

Method

Fig. 1 Fig. 2a Fig. 2b Fig. 2c Fig. 2d Fig. 4 denat, tau denat, tau (ref. 36) Fig. 4c

vert. repl. v.r. v.r. v.r. v.r. v.r. v.r. v.r. v.r.

14.5 +-0.9

(n = 6)

72.7.+ 6

(n = 5)

Fig. 3b

v.r.

14.1±2.6 17.5 +- 1.3

(n = 5) (n = 6)

81.4± 6.5 82.7+ 5

(n = 4) (n = 6)

v.r.

(n = 6) (n = 15) (n = 17)

14.9 5:2 14.1 +__0.7 16.8-+ 1.2

(n = 5) (n = 13) (n = 11)

79.8 ± 22 68.8+ 7.4 81.2± 8.3

(n = 6) (n = 16) (n = 13)

(n = 13) (n = 7)

21.7-+4 17.7.+3 20-25 25 18-22 24 ± 3.1

(n = 12) (n = 7)

68.6.+ 11.6 62.9.+ 12 80.1 +_14.5 80 70-80 65-80

(n = 11) (n = 7)

Fig. 3c (ref. 52) Fig. 2b Fig. 5a Fig. 5b (ref. 47) Fig. 4a Fig. 4b Fig. 4c (ref. 5) (ref. 19) (ref. 52) (ref. 53) Fig. 1 (ref. 27)

(n = 7)

10-20

(n = 4)

30-35

70-85

negative stain n.s. n.s. n.s. n.s. n.s. n.s. thin section t.s. t.s. t.s.

* All vertical replication T and W measurements were reduced by 0.6 nm or 0.7 nm for a 0.98 nm or a 1.14 Pt-C film thickness 34.

tion estimates of positive stain enlargement cannot be derived theoretically but must be derived empirically since stain concentration, period of staining, the sequence of stain application and staining temperature can change the total stain accumulation t4'38. The vertical replication and negative stain values of the widths were combined to estimate an average T and W for the

PHF (see Table II, column, Average mean ( A ) . . . ) . These values when subtracted from the thin section T and W values resulted in ATstai n -~ 6.6 nm and AWstai n = 9.5 nm. Since average stain enlargement values should be equal, the average T and W values used in the subtraction were examined. In vertical replication T can have values of 2.1-2.7 nm (n = 6 ) , 4.9 nm

TABLE II

Average of PHF measurements in Table I, by method PHF dimensions *

T(nm) W(nm) L (nm)

Vertical replication (v.r.)

Negative staining (n.s.)

Average mean (A) of corrected v,r. mean and n.s. mean

mean Pt-C coated

mean corrected i

mean (no correction needed)

mean

mean (mrs) with stain

estimate of stain mean thickness (mrs - A) 2 (mrs - 9.5 nm)

5.6+_3.1 15.5_+0.6 74.+8.5

4.9_+3.1 14.8-+0.6 74.+8.5

5.8+1.8 17.9-+3.2 74.9.+7.9

5.2+2.6 16.2-+2.7 74.4+8.0

11.8.+4.3 25.7.+6.0 75.7-+7.3

(6.6) (9.5) -

* The standard deviation was used as the uncertainty in the means, i The Pt-C coated means were reduced by 0.7 nm (see footnote in Table I). The sum of the positve stain deposits on both sides of the PHF.

Thin sectioning (t.s.).

2.3_+4.3 16.2.+6.0 75.7-+7.3

\

a

b

6

Fig. 2c (continued). ( n = 1), 7 . 1 - 7 . 7 n m ( n = 2), a n d 9 . 6 - 9 . 8 n m ( n = 2),

p r o t e a s e d i g e s t i o n , t a u o n t h e P H F s u r f a c e m,12'23'24~4~.

w h e r e a s W a p p e a r s to have only o n e value. If w e use

H i g h m a g n i f i c a t i o n i m a g e s in F i g s . 3b a n d 4 b a n d t h e i r

9.5 n m as t h e a v e r a g e s t a i n e n l a r g e m e n t t h e n t h e last

d r a w i n g s in Figs. 3 d a n d

c o l u m n in T a b l e II gives a c o r r e c t e d T v a l u e o f 2.3 n m ,

surfaces covered with

4d s h o w d i s o r d e r e d

~ 1.0 n m

strands

and

PHF some

which coincides with the most frequent vertical replica-

~ 0.4 n m s t r a n d s , t h e size o f n o r m a l a n d d e n a t u r e d t a u

tion

m o n o m e r 35. T h e p r o t o f i l a m e n t s t r u c t u r e s in Fig. 4a a r e

T

value

of

~ 2.4 n m .

The

corrected

average

d i m e n s i o n s of positively s t a i n e d P H F within thin sec-

also c o m p o s e d

tioned NFT

were

a r e L = 75.7 + 7.3 n m , T = 2.3 _+ 4.3 n m ,

a n d W = 16.2 + 6.0 n m .

of these

briefly washed

on

~ 1.0 n m s t r a n d s . T h e P H F a filter with distilled water

before they were freeze-dried and vertically replicated. The distilled water washing could scramble the surface

DISCUSSION

order of the tau monomer-like

~ 1.0 n m s t r a n d s b u t

w e c o n s i d e r t h i s u n l i k e l y s i n c e ~ 2.1 n m t r i p l e - s t r a n d e d

The dimensions, morphology and substructure of PHF T h e P H F h a v e b e e n s h o w n by p r o t e a s e d i g e s t i o n t o contain tau within the PHF

a n d , in t h e a b s e n c e o f

l e f t - h a n d helical tau p o l y m e r s w e r e visualized with this s a m e t r e a t m e n t 35. F u r t h e r m o r e ,

molecules of known

s t r u c t u r e , s u c h as t h e 1.33 n m h e l i x o f p e c t i n a n d t h e B

Fig. 3. a: a PHF compared to a ~ 2.1 nm tau-polymer-like filament. This PHF has a right-handed helical turn of L = 78+3 nm with an average width of 14/ 14.5+1.2 nm (15.2_+1.2 nm with a 0.7 nm Pt-C coating) at the center of the helical turn. The average thin region width is T = 9.8 + 0.2 nm (10.5 + 0.2 nm with a (1.7 nm Pt-C coating). A ~ 2.1 nm tau-polymer-like filament (2.8 nm with a 0.7 nm Pt-C coating) showing a left-hand twisted substructure crosses the PI-tF toward the top of the figure, b: a PHF compared to a ~ 2.1 nm tau-polymer-like filament at 1,930,000 magnification. The image area that includes this PHF surface is shown in Fig. 2d (1.1 nm thick Pt-C film). The tau-polymer-like filament crosses the PHF at the top of Figs. 2d and 3a. A pair of arrows points to one of the 1.0 nm diameter strands of the tau-like filament (1.7 nm with a 0.7 nm Pt-C coating). Two arrows mark similar 1.0 nm strands within the PHF surface (1.7 nm with a 0.7 nm Pt-C coating). There are also thinner strands within the within the surface of ~(I.4nm(1.0-1.1 n m w i t h a 0 . 7 n m Pt-Ccoating) but the 1.0 nm diameter strands are more frequent. The ~ 1.0 and 0.4 nm strands within the PHF are highly disordered, c: surface of mica coated with 1.0 nm of Pt-C. The Pt-C film shows structures at the 0.5-0.6 nm diameter level in this image 34. The strands shown in b are 2-3 times larger than 0.5-0.6 nm chain-like structures in the 1.0 nm thick Pt-C film. d: drawing of b.

,.J

form helix of double stranded D N A have also been visualized with this methodology 34. Nor can the appearance of the PHF surfaces be a replication artifact since vertical replication has shown single chain poly (1-tctradecene sulfone) helices and poly (cyclohexene sulfone) with no helices as predicted by low frequency dielectric loss experiments. This work has also shown the presence of side-chains on the poly (1-tetradecene sulfone), for the first time, and there absence in poly (cyclohexene sulfone) which has no side-chains. In addition this work has demonstrated that subtracting 0.4 nm from the Pt-C film thickness and applying this correction to the replicated polysulfones puts the polymer chain diameters within 0.05 nm of the diameters predicted by computer models ~v. The PHF dimensions measured in vertically replicated tangles, in negatively stained or in thin sectioned preparations, have roughly the same helical turn period (Table I|) as the thin sectioned PHF first reported by Kidd v~. The thin region,T = ~ 10 nm, and wide region, W - ~ 25 nm, dimensions which were not corrected for the positive stain deposition, are much larger than the average vertical replication measurements, the negative staining measurements, or the average of these two

methods (Table II). Measurements on the original images of PHF, assumed that the PHF had a periodic morphology and therefore a periodic substructure ~9'2°'s3. However, vertically replicated PHF in tangles has produced thin region T values of four different sizes. Although these T values appear in multiples of ~ 2.4 nm, there is no evidence for an organized ~ 2.4 nm protofilament or subunit structure in the P H F (Figs. 1-5). The observation that PHF's substructure is disordered suggests that PHF are not a periodic structure. To our knowledge, no one has ever reported a periodic filament without an ordered substructure. The PHF appear to be thin helical ribbons with no consistent evidence (Figs. 1-5) in our pictures for a paired helical filament morphology. We have estimated the average stain accumulation for the thin section PHF width measurements as 9.5 nm. After correction for stain enlargement, the PHF dimensions are L = 75.7 + 7.3 nm, T = 2.3 + 4.3 nm, and W = 16.2 + 6.0 nm. These dimensions are no longer compatible with the periodic paired helical filament model ~ or the subunit or protofilament models based on a periodic PHF model 21'45'47"4~. Since the most frequently occuring thin region measurement in vertically

Fig. 4a.

d

Fig. 4. a: a ~ 2.1 n m t a u - p o l y m e r - l i k e f i l a m e n t exiting the surface of a P H F . Two r i g h t - h a n d twisted P H F are shown e m a n a t i n g from the u p p e r left c o r n e r of this image. T h e u p p e r P H F turns sharply t o w a r d the u p p e r right corner. This P H F has a r i g h t - h a n d e d helical turn and d i m e n s i o n s of L = 6 4 + 7 nm, W = 15.1 +_0.9 n m (15.8+_0.9 with a (1.7 nm Pt-C coating) and T = 7.7___0.8 n m ( 8 . 4 + 0 . 8 with a 0.7 n m Pt-C coating). In the u p p e r left c o r n e r of the figure, the lower P H F splits unevenly, rejoins itself, and c o n t i n u e s t o w a r d the b o t t o m of the figure. This short i r r e g u l a r t e a r in the f i l a m e n t surface d o e s not qualify as a d o u b l e helical f i l a m e n t structure. Two ~ 2.1 n m f i l a m e n t s (2.8 n m with 0.7 n m of a d d e d Pt-C) showing a l e f t - h a n d twisted s u b s t r u c t u r e e m a n a t e from the u p p e r PHF. (This P H F surface is e n l a r g e d in b.) T h e ~ 2.1 n m t a u - p o l y m e r - l i k e filament m a r k e d with small arrows p o i n t i n g a l o n g its axis a p p e a r s to e n t e r the P H F s t r u c t u r e to r e - e m e r g e close to the two arrows that m a r k the filament d i a m e t e r of 2.1 n m (2.8 nm with 0.7 n m of a d d e d Pt-C). T h e g r o u p of p a r a l l e l arrows m a r k the ~ 2.1 nm f i l a m e n t with a l e f t - h a n d e d s u b s t r u c t u r e c o m p o s e d of 1 . 0 + 0 . 2 nm s t r a n d s (1.7 n m with 0.7 nm of Pt-C). A l t h o u g h p r o t o f i l a m e n t - l i k e divisions along the u p p e r P H F axis a p p e a r at the u p p e r right (2.3-3.8 nm wide), these divisions a p p e a r i n f r e q u e n t l y and are t h e m s e l v e s c o m p o s e d of 1.0 nm strands. The P H F do not contain ~ 2.1 n m t a u - p o l y m e r - l i k e f i l a m e n t s as a r e g u l a r part of t h e i r structure, b: a ~ 2.1 nm t a u - p o l y m e r - l i k e filament exiting the surface of a P H F at 2,000,000 magnification. This P H F surface and t a u - p o l y m e r - l i k e f i l a m e n t m a r k e d with 2.1 nm (2.8 nm with 0.7 nm of a d d e d Pt-C) in a is shown here at h i g h e r m a g n i f i c a t i o n (1.1 n m thick Pt-C film). This same p o l y m e r i c tau-like f i l a m e n t c o m e s out at the right side of the P H F with one of its ~ 1.0 n m s t r a n d s m a r k e d by arrows (1.7 nm with a 0.7 n m Pt-C coating). D o u b l e and single arrows also m a r k similar 1.0 nm s t r a n d s within the P H F surface (1.7 nm with a (].7 nm Pt-C coating). T h e r e are also a few ~ 0.4 nm s t r a n d s (1.0 1.1 nm with a [~.7 nm Pt-C coating) within the surface, but the ~ 1.0 nm d i a m e t e r s t r a n d s are m o r e frequent. T h e s t r a n d s within this P H F are highly d i s o r d e r e d as they were in Fig. 3b. c: surface of mica c o a t e d with 1.0 n m of Pt-C. T h e Pt-C film shows c h a i n - l i k e s t r u c t u r e s at the 11.5-(/.6 nm d i a m e t e r level ( R u b e n , 1989). The s t r a n d s shown in b are 2 - 3 times l a r g e r t h a n the 0.5 0.6 n m c h a i n - l i k e s t r u c t u r e s in the 1.0 n m thick Pt-C film. d: d r a w i n g of b.

10 replicated PHF is also T = ~ 2.4 nm (Table I), the PHF more closely resemble thin (2.1-7.1 nm thick) helical ribbons.

Can tau polymer filaments be the precursor of PHF? We have only seen normal, ~ 1.0 nm, and probably some denatured, ~ 0.4 + 0.2 nm, tau monomer strands in the surface of the PHF and we have not observed either amyloid fibers 36 or ubiquitin. The PHF have been shown to stain with antibodies to both tau and ubiquitin3,7,8,10 12,16,22,30,31,49,55,56. Tau has been shown to be the major subunit of P H F 1°'1s'24. Ubiquitin's association with NFT is minor and usually occurs in the later stages of tangle formation 3'4. Ubiquitin is a single 76 amino acid chain of 8,565 daltons which forms a globular protein recognizable by its dimensions of ~ 3 n m × ~ 4 nm × ~ 5 nm 43. The strong concern that extraneous cytoskeletal proteins adhere to P H F and associate on its external surface has led to removal of the PHF outer surface with proteinase digestion. The

undigested PHF core as well as the PHF surface has been shown to contain t a u 10'12'23'24'49 in abnormally phosphorylated f o r m s 11'16'18"24. Evidence for the presence of neurofilaments, MAP-2, or other cytoskeletal proteins in PHF is weak or negative 3'4. In addition, it has recently been shown that tau forms ~2.1 nm diameter triple-stranded left-hand helical polymers composed of three ~ 1.0 nm (diameter) x ~ 62.4 nm (long) strands 35. Each strand is capable of forming two or one disulfide linkages through the cysteines at amino acid 291 a n d / o r 322, depending on whether the tau isoform contains four or three microtubule binding region repeats in a primary sequence of 441 or fewer amino acids 9. These strands probably form, at most, a single link to an adjacent strand with the remainder of each strand hydrophobically associated with the other two. The triple-stranded filament segments probably join N to C terminal by opposite charge interaction making this polymer many times the length of a monomer unit or a filament segment 35. A long poly-

Fig. 5. ~ 2.1 n m t r i p l e - s t r a n d e d left-hand helical p o l y m e r i c tau f i l a m e n t s h e a t e d to 9t°C. I s o l a t e d bovine tau p r o t e i n was t r a p p e d on a 0.1 ~ m Millipore filter and w a s h e d with distilled water. O n e set of these s a m p l e s was f r e e z e - d r i e d and vertically r e p l i c a t e d with 1.04 nm Pt-C and b a c k e d with 13 nm of e v a p o r a t e d c a r b o n (c). In a and b the tau s a m p l e s w e r e h e a t e d to 91°C on a w a t e r b a t h for 15 rain, b l o t t e d and t h e n frozen. This s a m p l e was p r e p a r e d as above with a 1.02 nm Pt-C film a n d b a c k e d with 13 n m c a r b o n film. T h e s e i m a g e s of tau suggest that the d e n a t u r a t i o n of ~ 8(10 nm-long single native tau f i l a m e n t s (c) can e x p a n d their width to the size r a n g e of the PHF. Only two regions in b a p p e a r to have the average helical turn length of P H F . The wides are also roughly 15.4 n m and 16.4 n m (16 nm and 17 n m wide with a 0.6 nm Pt-C coating) or e q u i v a l e n t to the wide regions in PHF. T h e f i l a m e n t s in a a n d b are 636 nm and 818 nm in length, respectively. T h e r e are also regions in a and b w h e r e the width a v e r a g e s 2.2 nm, 4.4 nm, 8.4 nm and 10.4 nm, e q u i v a l e n t to P H F thin regions widths, T (see T a b l e I: add 0.6 nm for the Pt-C c o a t e d d i a m e t e r s in a and b). T h e r e are also wide region widths, W, of 15.4 nm, 16.6 nm, 18.4 nm, 20.4 n m and 28.4 n m which are similar to those found in the P H F (add (1.6 n m for the Pt-C c o a t e d size in a and b).

11 meric tau filament of this type could easily be denatured into associated ~ 1.0 nm tau monomer units or 0.4 + 0.2 nm diameter amino acid chains from tau monomers. We have attempted to show that bovine tau polymer filaments attached to a Millipore filter can be partially heat denatured (91°C for 15 min) into filaments with some PHF-like characteristics. This approach confines the mechanism of formation of P H F to the direct denaturation of a single tau polymer filament. Images of two partially denatured filaments are shown in Fig. 5a,b and two regions of the filament in Fig. 5b were included in Table I (T = 3.2 and 3.6 nm, W = 17 and 16 nm, L = 56 and 45 nm). The heat treatment of tau has generated some but not all of the features of PHF. The wide regions, W, fall within the range of widths for vertically replicated and negatively stained PHF. Heat denatured thin region widths, T, are ~ 2.2 nm, ~ 4.4 nm, ~ 8.4 nm and ~ 10.4 nm (corrected for the Pt-C coat of 0.6 nm) and are equivalent to those measured in the PHF. The wide regions are shorter or fall within the range of helical turn lengths, L, for the PHF. These partially denatured tau filaments have no obvious twist or helicity. Motejo de Garcini et al. have treated porcine tau with 8 M urea for three days at room temperature and have produced both right- and left-handed PHF-like filaments with a helical turn period of L = 70 nm, W = 20-25 nm and T = ~ 10 nm 28'29. Relatively uniform unmodulated filaments of 10.6 nm, 13.8 nm and 15 nm diameters were also denaturation products of tau. Even previously reported negatively stained NFT isolates show PHF, straight filaments (width: 7 nm and 10 nm), and PHF-like filaments which merge with straight filaments (see Fig. 4 ) 47 . The denaturation experiments suggest that it should be possible to produce P H F as well as generate the variety of straight filamentous structures seen in N F T 36. In the absence of glutaraldehyde fixation, it has been shown that the denaturing effect of osmium tetroxide on N F T produces P H F 32. When osmium tetroxide or glutaraldehyde followed by osmium tetroxide react with native proteins in solution or in membranes they frequently denature the native structure 25, although prior aldehyde fixation more effectively prevents protein extraction or major structural rearrangemerits ~4. Papasozomenos has reported that osmium tetroxide treated Alzheimer brain tissue without prior glutaraldehyde fixation produces N F T that are almost exclusively P H F but with glutaraldehyde fixation this was prevented 32. Tissue fixation regimes that employ osmium tetroxide without aldehyde fixation even for short time periods extract 5 - 2 3 % of the protein 6'26. It

has been shown that osmium tetroxide can liberate ammonia from proteins, oxidize a number of amino acid side chains as well as oxidize disulfide bridges 13. The first P H F observations were made in the absence of glutaraldehyde fixation with a 1% osmium tetroxide or a 0.6% pemanganate treatment 19, where permanganate fixation is even more denaturing than straight osmium fixation 25. If the P H F are a denaturation product of an integral tau polymer filament in NFT, then clearly osmium fixation without glutaraldehyde treatment could produce PHF. The relationship between denatured tau polymer and P H F also raises the possibility that all isolation procedures which employ strong detergent (sodium dodecyl sulfate, SDS) a n d / o r sonication could be producing P H F as well as isolating PHF. Perhaps this explains why the yield of P H F is higher in the presence of detergent 15'19'39'4°'47, why NFT isolated with SDS detergent are almost exclusively P H F 33, and why N F T isolated in the absence of detergent are heterogeneous with the major portion of the NFT composed of ~ 2.1 nm tau polymer filaments 36. We do not mean to imply that all P H F are created artificially. Intracellular proteinase activity could create P H F but probably not in the same high frequency that has been reported in the NFT. The most intriguing feature of the Alzheimer filaments is their helical twist. Isolated P H F have been reported to have both left-handed 45'47 and right-handed twist 5, and thin sectioned material has been reported to have left-handed and some right-handed P H F 54. The Alzheimer filaments in Figs. 1-5 all have righthanded helicity. If P H F are denatured abnormally phosphorylated tau polymer filaments, we could expect the triple-stranded left-hand helical polymer filament to unwind internally even while associated with other filaments. The new denatured filament's helicity could be left-hand helical, non-helical, or right-hand helical depending on how much of the original left-hand helical tau polymer was unwound. The helical nature, the partially regular filament morphology, and the nonperiodically aggregated 1.0 nm and 0.4 nm strands in P H F could be attributed to the denaturation of ~ 2.1 nm tau triple-stranded left-hand helical polymers. We do not know if more than one tau polymer is the precursor of a single PHF. It is clear, however, that if single tau polymers are the P H F precursor then the denatured ~ 2.1 nm tau polymer would have to associate laterally with itself and shorten substantially as it collapsed to form the generally wider PHF. P H F formation from tau polymer denaturation is too complex to model at this time. The variability in P H F probably depends on the

12 conditions, kinetics, and extent of tau's denaturation, as well as on tau's primary amino acid sequence and on its phosphorylation state. Twisted PHF-like filaments found in chronic alcohol administered rats (L = 35 nm, W = 15-20 nm, T = 10 nm) have a much shorter helical turn but are otherwise similar to human P H F 44. Brain tissue from aged Rhesus monkeys also contains PHFlike filaments with a shorter helical turn period, L = 50 nm, but with W and T values similar to human P H F 51. If P H F are a denaturation product of human tau polymer, it is unlikely that denatured rat or monkey tau would have exactly the same helical turn period. It was previously accepted that P H F ' s morphology was periodic and unique and that its absence in animal brains should rule out animal models for Alzheimer's disease. The difference in animal and human P H F morphology should no longer prevent the development of Alzheimer N F T animal model systems. Acknowledgements. G.C.R. wishes to thank GeoM Co. for its support and the Dartmouth Rippel Electron Microscope Facility for the use of the JEM 100CX and the Balzers 300. Preparations of NFT were made by Ms. T. Zaidi and Dr. N. Ali in K. Iqbal's lab. Alzheimer disease brains were obtained from the Human Neurospecimen Bank (Dr. G.G. Glenner), University of California, San Diego, CA and from the Netherlands Brain Bank for Alzheimer's Disease (Drs. R. Ravid and D. Swaab), Amsterdam, Netherlands. Support for this work was provided in part by New York State Office of Mental Retardation and Developmental Disabilities, NIH Grants AG05892, AG04220, AG08076 and NS18105, and a grant from the Alzheimer's Research Pi-ogram of the American Health Assistance Foundation, Rockville, MD.

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