Quantitative solubilization and analysis of insoluble paired helical filaments from alzheimer disease

BRAIN RESEARCH

ELSEVIER Brain Research 717 (1996) 99-108

R e s e a r c h r e p o r t

Quantitative solubilization and analysis of insoluble paired helical filaments from Alzheimer disease

M a r k A. S m i t h a, S a n d r a L. S i e d l a k a P e g g y L. R i c h e y a, R a m a n a k o p p a H . N a g a r a j a, A k e E l h a m m e r b, G e o r g e P e r r y a, *

a Institute of Pathology, Division of Neuropathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106-2622, USA b Upjohn Company, Kalamazoo, M149001, USA

Accepted 14 November 1995

Abstract

In this study, we evaluate the ability of several solvents to solubilize insoluble paired helical filaments (PHF) of Alzheimer disease. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents of PHF. Using sequential categories of protein solvent to analyze insoluble PHF, only alkali or exhaustive proteolysis are effective in completely solubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosphorylation of PHF with hydrofluoric acid does not affect PHF solubility. These findings suggest that the 'hyperphos- phorylation' of PHF proteins is not responsible for PHF insolubility. However the in vitro glycation of z generates PHF that are insoluble in SDS and soluble in alkali. These findings suggest that protein crosslinks, including advanced glycation endproduct-derived crosslinks which were recently described in Alzheimer disease, play a major role in effecting PHF insolubility in vivo.

Keywords: Alzheimer disease; Crosslink; Glycation; Neurofibrillary pathology; Paired helical filament; Solubility

1. Introduct ion

The paired helical filaments (PHF) of neurofibrillary tangles are the most striking intraneuronal change seen within the brains of patients with Alzheimer disease (AD). However, despite intense efforts to understand the molecular composition of PHF, quantitative biochemical analyses are severely hampered by the extreme insolubility of PHF [51] and by difficulties obtaining a homogeneous PHF fraction. To date, all of the published studies on the biochemical composition of insoluble PHF (SDS-insoluble) are qualitative and have provided little or no quantitative data on the proportion of material assayed. Neverthe- less, biochemical, immunochemical , amino acid composition and protein sequencing techniques in studies of insoluble PHF demonstrate the presence of ~- and ubiquitin [19,38,43,65].

While the PHF of neurofibrillary tangles have the previ-

Corresponding author. Fax: (1) (216) 368-8964. E-mail: gxp7 @ po.cwru.edu

0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDIOOO6-8993(95)OI473-X

ously described properties, a separate, though probably related, soluble PHF (A68, PHF-z or SDS-soluble PHF) were biochemically isolated [15,17]. The heterogeneous composition of insoluble PHF contrasts with soluble PHF, which are composed entirely of highly phosphorylated z isoforms [17,26,31,40,66]. The presence of two types of PHF leads to the proposition that 'abnormal phosphorylation' of z is the first step in PHF formation and that soluble PHF are the progenitors of insoluble PHF [12,14,18]. However, transformation of soluble PHF to insoluble PHF is unlikely to be solely mediated by phosphorylation, since in vitro phosphorylation of normal z to the AD-like state has little, if any, effect upon solubility [201.

In addition to phosphorylation, three other post-transla- tional modifications described in AD, racemization [8], isomerization [42] and ubiquitination [38,43], are also unlikely to have any effect on the solubility properties of PHF protein. However, another class of posttranslational modification, that would account for the insolubility of PHF, advanced glycation endproducts (AGE), were recently described in AD [30,58,63,67]. Protein modification

100 M.A. Smith et a l . / Brain Research 717 (1996) 99-108

by AGE is associated with crosslink formation leading to decreased solubility and increased resistance to proteolysis [37,49].

The present study quantitatively assesses the solubility properties, defined as protein in solution and reduction in fraction volume, of insoluble PHF in a series of solvents and provides evidence that PHF proteins are covalently crosslinked. Insoluble PHF are resistant to solubilization in SDS, formic acid, 4 M urea and 8 M guanidine-HC1, but are solubilized by extraction with alkali or prolonged proteolysis. These solubility properties can be conferred to soluble PHF by in vitro glycation with reducing sugars suggesting that non-enzymatic glycation could play a role in the conversion of soluble PHF to insoluble PHF. To- gether, our data support the idea that the biochemical properties of insoluble PHF may be mediated by AGE-dependent protein crosslinks.

2. Materials and methods

2.1. Insoluble PHF and control fractions

Hippocampus and temporal cortex from individual cases, with meninges and white matter removed, were homogenized in 5 vols. of 50 mM Tris-HC1, pH 7.6 containing 0.1 g S D S / g wet tissue. The homogenate was centrifuged at 10,000 X g for 4 h at 22°C, the supernatant removed, and the pellet rehomogenized in 1% S D S / 1 0 % sucrose /50 mM Tris-HCl, pH 7.6 (Buffer A). After centrifugation at 25,000 X g for 1 h, the pellet was again homogenized in 1% SDS/Buf fe r A and layered over a gradient of 12.5 ml each of 1.0 M, 1.2 M and 2.0 M sucrose/Buffer A. Following centrifugation for 1 h at 72,000 X g the insoluble PHF fraction at the 1.2-2.0 M interface was used in further experiments.

PHF fractions were prepared from Alzheimer disease ( n = l l ; mean 75.2 years; range 61-83) and identical control fractions were prepared from young controls (n = 6; mean 39.2 years; range 17-53) and old non-demented aged controls (n = 4, mean 68.0 years; range 65-73). A minimum of three cases were individually used for each portion of the study, and representative data are shown.

2.2. Soluble PHF

Following the method of Lee et al. [31], the grey matter of hippocampus and temporal cortex from cases (n = 3) of Alzheimer disease was homogenized in l0 vols. of Buffer B (0.8 M NaC1, 10% sucrose, 50 mM Tris-HC1, pH 7.0) and centrifuged at 27,000 × g for 20 rain at 4°C. The pellet was re-extracted in Buffer B and recentrifuged as previous. The supernatants from both centrifugations were combined, made to 1% Sarcosyl, incubated at 22°C for 30 min, centrifuged at 100,000 × g for 1 h at 15°C and the pellet used in further experiments.

2.3. Other proteins

Heat stable microtubule-associated proteins were prepared from bovine brain microtubules [24]. ~- was purified from heat-stable microtubule-associated protein prepara- tions [32]. Neurofilaments were prepared from human peripheral nerve [53]. Amyloid-/3, a synthetic 42mer peptide, was prepared as previously described [4].

2.4. Antibody characterization

The following well characterized antibodies were used: monoclonal antibodies (mAbs) to each of the three neurofilament subunits (Biomakor, Israel) [52]; antisera and mAbs to c~- and /3-tubulin [44]; two different antisera to P-component [27]; affinity purified antiserum to 7 [45]; Alz-50, a mAb to a modified form of ~- [66]; 5E2, a mAb to ~- [10]; Tau-1, a mAb to a dephosphorylated epitope of ~- [2]; affinity purified antiserum to amyloid-/3 [4]; affinity purified antiserum to tropomyosin [11]; affinity purified antiserum to ubiquitin [34].

2.5. Extraction agents

The sequential extraction agents were divided into six categories. (i) Detergent Treatment: 1% SDS; (ii) Disulfide Bond Cleavage: 1% S D S / 1 % /3-mercaptoethanol (/3 ME); (iii) Ionic Disruption: 5 M NaCI or 0.1 M EDTA; (iv) Denaturants: 8 M urea, 6 M guanidine-HC1 or 70% formic acid; (v) Cold Alkali Treatment: 0.2 or 1 M NaOH at 37°C; (vi) Hot Alkali Treatment: 1 M NaOH at 90°C.

2.6. Extraction procedure

Extraction with each agent was performed for 10 min at room temperature with the exception of the alkali treat- merits which were at 37°C or 90°C. After each extraction, the sample was centrifuged at 16,000 X g for 30 min at 4°C and the supernatant analyzed by 10% polyacrylamide gel electrophoresis (SDS-PAGE) according to standard procedures [29] and the pellet was then re-extracted in the same agent. Alkali extractions were neutralized with 1 M Tris-HC1 following centrifugation. In initial experiments no significant differences were noted if samples were centrifuged at 170,000 × g for 1 h.

Extracted proteins were defined as solubilized if present in the supernatant following centrifugation and were able to enter the running gel of SDS-PAGE. Protein was quanti- tared with an LKB Laser Gel Scan (Pharmacia-LKB) by densitometric scanning of SDS-PAGE stained with either Coomassie brilliant blue R250 or silver [39]. The entire running gel, but not the stacking gel containing insoluble material unable to enter the gel, was used for densitometric measurements. Densitometric readings were expressed as absorbance units multiplied by area (ram2), and data shown

M.A. Smith et al./Brain Research 717 (1996) 99 108 101

are intentionally uncorrected for baseline artifacts produced by each solvent.

Chemical treatment often resulted in absorbance readings with no addition of protein. To resolve this problem we sequentially performed each extraction at least five times (in preliminary experiments up to ten extractions were used for some agents). The rationale for our approach is that artifactual absorbances remain constant for all extracts whereas readings of extracted proteins are greatest in the initial extracts. For this reason our data are represented without correcting for baseline artifacts. In practice, we found that essentially all possible extractable protein was removed by the third treatment for the time and agents used.

2.7. Determination of PHF fraction volume

Quantitation of the reduction in fraction volume following extraction was also used as a measure of solubilization. After each extraction the PHF were drawn into capillary tubes (1 mm i.d.) and centrifuged at 2500 × g for 30 rain. Quantification of 'volume' was made by measuring the height of the pellet with the reticule of a magnifier (Bausch and Lomb, × 7, calibrated to 0.1 mm) and compared to the height of the pellet obtained from the original PHF fraction.

2.12. Phosphate assay

Phosphate detection was by standard procedures [1,48]. Inorganic phosphate standards were prepared using anhy- drous KH2PO 4 in 0.01 N H2SO 4 (1:1 w/v) .

2.13. Proteolysis treatment

Proteinase K or Pronase (1 mg/ml) were incubated with the insoluble-PHF at 55°C for 10 min and analyzed as described above.

2.14. Electron microscopy

Fractions were fixed in 1% glutaraldehyde and post- fixed in 1% OsO 4 for 1 h, dehydrated in graded ethanol followed by acetone and embedded in Spurr's medium. Sections were cut parallel to the direction of centrifugation and the entire depth of the pellet examined following contrasting with uranyl acetate and lead citrate.

2.15. Carbohydrate analysis

Total sugar was determined as previously described [6,22,62].

2.8. Amino acid analysis

Specimens were hydrolyzed in 6 N HC1 with 80 mM /3 ME in sealed glass tubes under argon at 115°C for 18 h. Samples were dried and dissolved in citrate buffer pH 2.2 and applied to a Beckman l l9CL amino acid analyzer [36]. The glycine peak was used since it is not affected by NaOH.

2.9. Nonenzymatic glycation of soluble PHF

Soluble PHF (175 /_~g) was incubated in 100 /~1 of 0.25 M sugar (DL-glyceraldehyde, threose or ribose) in 0.1 M phosphate buffer, pH 7.4, at 37°C for 3 days and used for further analysis.

2.10. Immunoblotting

Proteins separated by SDS-PAGE were electroblotted onto Immobilon-P (Millipore) and immunostained by using standard procedures [29,61].

2.11. Dephosphorylation of PHF protein

Insoluble PHF were treated in 50% hydrofluoric acid (HF) for 48 h at room temperature. Excess HF was removed by Speed-Vac centrifugation.

3. Results

Two complementary measures were used to objectively and quantitatively assess the amount of protein solubilized: solubilization of protein and reduction of fraction volume. These two approaches are chosen because they are very reproducible, precise and not dependent on pure PHF fractions. Densitometric evaluation of five sequential extractions with each agent showed that most protein was solubilized in the first two or three extractions when either Coomassie blue or silver stains are used, beyond this point the values return to a baseline produced by artifacts of the solvent (Figs. 1 and 2). Fig. 1 shows the results of densitometry of the sequential extracts on SDS-PAGE stained with silver. Extraction in 1% SDS, the buffer employed in preparing the insoluble PHF fraction, con- firmed that all elements soluble in SDS were removed by the isolation procedure (Fig. l i). Surprisingly, following SDS//3ME extraction (Fig. lii), no further proteins are extracted by the denaturants urea, guanidine or formic acid (Fig. liii, iv). The results of replicated multiple extractions (Fig. 2) of the most potent extracting agents, but in the case stained with Coomassie blue, show most of the material is removed in the first two to three extractions. This finding contrasts with previous reports showing par- tial solubilization of PHF in formic acid or guanidine [35,47]. The extracted proteins migrated at all molecular masses with variable amounts of discrete protein bands (Fig. 3).

102 M.A. Smith et al. / Brain Research 717 (1996) 99-108

14 i/ii

12

10

8 ,<

o O9

iii v/vi

GO LU LU "0

o~ ~ ua "~ ._o ~ z z

03 ~ o c5

Ex t rac t i on A g e n t s

Fig. 1. Densitometric analysis of 5 sequential, left to right, extracts of a insoluble PHF fraction with agents found to be effective in extracting protein analyzed by SDS-PAGE followed by silver staining. Most protein is solubilized in the first 2-3 extractions. Each panel shows a different category of extracting agent, i.e., it had previously been extracted with the reagent preceding it in the hierarchy (i-vi, see Section 2). In (i / i i) /3 ME, or even more effectively, SDS-/3 ME solubilized proteins. Follow- ing SDS-/3 ME extraction of the PHF; high salt, divalent chelation (iii) or denaturants (iv) were ineffective in solubilizing proteins. In contrast, NaOH treatment at 37°C and even again at 90°C was effective in extracting protein (v/vi). Extracted protein is expressed as histogram heights in units of absorbance multiplied by area (mm 2) ( n - 5 ; _+ S.E.M.).

O r i g i n a l

~B~- •

2os,. 116>- 9 7 ~ 6 6 ~'~ ~ 451~- ~

r 2 9 ~ -

B I

1 I I I I I I V V

: - i (

205]~

6 6 ~ -

451D~

2 9 ~

D ~? -/~s ~

1.2

1.0.

0.8

0.6

(.~ 0.4

0.2

0.0 1 2 3 4 5

SDS/[~ME 1 2 3 4 5

1.0M NaOH 37°C

Ex t rac t i ons A g e n t s

1 2 3 4 5 1.0M NaOH 90°C

Fig. 2. Densitometric analysis of five sequential, left to right, extracts of an insoluble PHF fraction with agents found to be effective in solubilizing protein analyzed by SDS-PAGE followed by Coomassie blue staining. Most protein is solubilized in the first 2-3 extractions. Solubilized protein is expressed as histogram heights in units of absorbance multiplied by area (ram2).

i~ii!i!iiii;i i~!ii!iiiiiii!:ii 29>,~

Fig. 3. Coomassie blue stained SDS-PAGE of the 5 sequential, left to right, extracts of an insoluble PHF fraction. A, original sample; B, SDS-/3ME extraction series; C, NaOH at 37°C extraction series; D, NaOH at 90°C extraction series. The major discrete bands shown in all extracts are between 60-64 kDa. Arrow, top of 4.5% PAGE stacking gel; large arrowhead, top of running gel; small arrows, molecular mass markers in kDa. The scanned area is shown by the lines on the far right.

E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n o f the i n s o l u b l e P H F

f r ac t i on f r o m A D c a s e s (Fig . 4), bu t no t f r o m the c o n t r o l

f r ac t i ons , s h o w e d a b u n d a n t P H F and va r i ab l e q u a n t i t i e s o f

b a s e m e n t m e m b r a n e and u n i d e n t i f i a b l e deb r i s , t he la t te r

a l so b e i n g p r e s e n t in the f r a c t i on p r e p a r e d f r o m con t ro l

bra in . A f t e r S D S / / 3 M E e x t r a c t i o n the f r ac t ion a p p e a r e d to

be qu i t e s i m i l a r to t he o r ig ina l , h o w e v e r , f o l l o w i n g alkal i

e x t r a c t i o n at 37°C t h e r e are r e l a t i ve ly f e w iden t i f i ab l e

M.A. Smith et al./Brain Research 717 (1996) 99-108 103

W h i l e the P H F f rac t ion l acked o l igosacchar ides , it con-

t a ined 6 - 9 - t i m e s ( w / w ) suc rose (a n o n r e d u c i n g sugar)

a cqu i r ed f r o m the pur i f i ca t ion buffer . Its a ssoc ia t ion is

b a s e d on a d h e r e n c e ra the r than by c o v a l e n t a t t a c h m e n t and

A A B C D E

205 ~ !

116>.- i i 6 6 ~

I

F

Fig. 4. Electron micrographs of SDS-/3ME extracted fraction showing PHF (insert).

PHF , and af te r a lkal i ex t r ac t ion at 90°C, e v e n less P H F

were noted. The d i a m e t e r o f the P H F f o l l o w i n g each

t r e a t m e n t s h o w e d no s ign i f i can t d i f f e rence ( T a b l e 1). In

cont ras t , the v o l u m e o f res idua l mate r ia l af ter each ext rac-

t ion was readi ly and r e p r o d u c i b l y r educed to neg l ig ib l e

v o l u m e s by alkal i ex t rac t ion .

W e f o u n d S D S / / 3 M E t r e a t m e n t o f P H F did not grea t ly

r educe the v o l u m e o f ma te r i a l ( re la t ive v o l u m e 9 6 % _+ 1.9),

h o w e v e r , i n c u b a t i o n wi th a lkal i at 37°C or 90°C r e d u c e d

the re la t ive v o l u m e o f i n so lub le ma te r i a l to 5 . 0 % _ 0.2

and 0 . 2 7 % _+ 0.28, r e spec t ive ly ( T a b l e 1). W i t h less than

1% of the f r ac t ion r e m a i n i n g , the bu lk o f P H F are so lubi -

l ized by alkali . A m i n o acid ana lys i s o f the so lub i l i zed

ma te r i a l s h o w e d that ove r 7 6 % of the p ro t e in is so lub i l i zed

by N a O H at 37°C.

Table l Summary of immunoblot and electron microscopic observations for insoluble PHF

PHF Fraction Residual Solubilized PHF Diameter volume proteins (nm _+ S.E.M.) (% _+ S.E.M.)

Original PHF 100% ~-, neurofilaments, 17.0 _+ 0.6 tubulin, P-component, ubiquitin

SDS//3ME 96.0_+1.9% ~- 18.3_+0.7 NaOH at 37°C 5.0_+0.2% ~- 18.8_+0.9 NaOH at 90°C 0.3_+0.3% 7 17.0_+0.6

Only ~- is released in extracts following SDS//3ME. Measurement of PHF filament diameter following extractions shows no significant differ- ence by non-parametric Student's t-test. However, this finding must be seen in the context of our residual volume-based assay showing that only a very small fraction of material remains following alkali treatment.

45>"

24>" !

B

116>~ !- 66~,-

24]=.-

i

C

116]=,~ 6 6 ~

I

L

[ t

Fig. 5. Immunoblot analysis of the proteins m supernatants obtained from insoluble PHF by agents effective in protein extraction: (A) SDS-/3ME extract; (B) NaOH at 37°C; and (C) NaOH at 90°C extract. The same antibodies were used with both the extract and pellet: antiserum to ~- (A); mAb to ~- (5E2) (B); mAb to /3-tubulin (C); antiserum to P-component (D); antiserum to ubiquitin (E); antiserum to amyloid /3 protein (F). While immunoreactivity was seen for ~-, tubulin, P-component, neurofilaments and ubiquitin in the SDS-/3ME supematants, only ~- was noted in the two alkali extracts. Arrow, top of 4.5% PAGE stacking gel; large arrowhead, top of running gel; small arrowhead, molecular mass in kDa.


is completely removed by treatment with amylase. Amy- lase treatment prior to further extraction did not change the relative residual volume measured after the various treatments.

Immunoblott ing (Table 1 and Fig. 5) shows the initial extracts with SDS-/3ME contained a variety of protein elements, including neurofilament protein, tubulin, ubiquitin, ~-- and P-component, whereas subsequent extraction with alkali at 37°C or 90°C only releases T-related proteins. Although many proteins were solubilized by S D S / / 3 M E , protein release was not correlated with a substantial decrease in fraction volume (Table 1). In contrast, alkali extraction resulted in greater protein extraction and volume decrease. This suggests proteins extracted by alkali are related to the bulk of insoluble PHF, whereas the SDS- /3 ME extracted proteins are either associated elements of PHF or a subclass of PHF which are soluble in SDS-/3 ME, but not in SDS. Analysis of fractions prepared in parallel from brains lacking neurofibrillary tangles show many of the same elements identified in the S D S / / 3 M E extracts (data not shown), supporting the view that the SDS-/3 ME extracts of PHF are principally associated elements. The low level of ~- immunoreactivity in the alkali extracts, compared to the amount of protein solubilized, could be the result of reduction in ~- epitopes by alkali (see below), blockage by glycation (see below) or that other components are distinct in insoluble PHF.

Alkal i could cause base-hydrolysis affecting the densitometric analysis or immunoblot identification of solubilized protein. Therefore, we treated purified neurofilament, ~- and amyloid-/3 protein with alkali as described for the solubilization protocol. Densitometric scanning of these purified proteins after alkali treatment for 10 rain at 37°C showed that the quantity of protein is reduced by 5.02% for neurofilaments and by 51.29% for A/3 and is unchanged for ~-. Moreover, discrete protein bands at the original molecular mass are still readily apparent on SDS- PAGE analysis as well as by immunoblott ing (results not shown).

Proteolysis with pronase, proteinase K and to a lesser extent HC1 (6 M HCI, 95°C, 12 h) reduce fraction volume as treatment with alkali, but differ from alkali treatment since no proteins are identifiable by SDS-PAGE analysis or immunoblots (data not shown). These findings suggests that alkali treatment is not solely solubilizing PHF due to peptide bond hydrolysis but may be affecting cleavage of another protein-based linkage.

One possibil i ty is alkali might dephosphorylate PHF. However, we found that the level of organic phosphate is essentially unchanged by the alkali treatments used here (Table 2). Further, complete dephosphorylation of PHF with hydrofluoric acid (Table 2) did not reduce the fraction volume or change subsequent solubility of the insoluble PHF in other solvents (results not shown). Together these data suggest that organic phosphate moieties present in PHF proteins are not responsible for insolubility.

Table 2 Quantity of phosphate associated with insoluble PHF following alkali extraction or hydrofluoric acid treatment

Treatment /~mol Phosphate/g protein ( + S.E.M.)

Original Organic 72.5+2.8 n 4 Inorganic 5.9 _+ 0.8 n = 4

1.0 M NaOH 37°C Organic 58.1 _+ 6.9 n = 9 Inorganic < 0.01 n = 8

l./) M NaOH 90°C Organic 64.3 + 8.6 n - 9 Inorganic < 0.01 n = 7

Hydrofluoric acid Organic < 0.01 n - 10 Inorganic 75.4 + 0.6 n - 2

While the latter treatment was effective in releasing all organic phosphate residues as inorganic phosphate, alkali does not release organic phosphate groups.

Advanced glycation end products (AGE), recently identified in PHF [58,63,67], confer insolubility to a variety of proteins. However ~- in soluble PHF seems a particularly appropriate model since it contains abundant lysines and is considered to be the precursor of insoluble PHF. To deter- mine if AGE could modify soluble PHF to mimic the solubility properties of insoluble PHF we chose to chemi- cally glycate soluble PHF protein by incubation with various reducing sugars: DL-glyceraldehyde, threose or ribose. Our data (Fig. 6) shows that soluble PHF incubated without sugar are readily soluble in S D S / / 3 M E , whereas soluble PHF incubated with either 0.25 M DL-glyceraldehyde, threose or ribose are relatively insoluble in S D S / / 3 M E , but are soluble in alkali. Therefore glycation alone can account for the differential solubility between insoluble PHF and soluble PHF. This further suggests

i LL.. "1- 13_

t -

t -

09 0_

100 - -

90

80

70

60

50

40

30

20

10 L Buffer 0.25M 0.25M Threose 0.25M Ribose

Glyceraldehyde

[] Untreated [] SDS/[3ME [] NaOH 37°C

Fig. 6. Volume analysis of soluble PHF protein incubated with different reducing sugars for 3 days at 37°C. Protein was extracted for 30 rain with buffer (untreated), SDS//3ME or 0.2 M NaOH at 37°C. Protein is completely solubilized by 0.2 M NaOH at 90°C, not shown.

M.A. Smith et al. / Brain Research 717 (1996) 99-108 105

glycation plays a role in the conversion of soluble PHF to insoluble PHF [30,58,67].

4. Discussion

In this study we quantitatively measured PHF solubilization by coupling a volume-based assay of the residual fraction together with densitometric measures of solubilized protein on SDS-PAGE. The power of this method is the highly reproducible quantitation and that it corresponds to standard assays such as amino acid analysis. We found that strong denaturants only extract associated proteins from PHF and that these protein components can also be removed by extensive SDS/ /3ME extraction. The complete solubilization of PHF requires alkali treatment or prolonged proteolysis, and this agrees with previous studies reporting the effects of alkali treatment on the structure of PHF [26,64].

The suggestion that phosphorylation is important for PHF insolubility [ 14,18] is not supported by our data, since complete dephosphorylation of PHF has no effect on solubility. Moreover it is not supported by the work of others. Transformation of 7 to the AD-like state by phosphorylation does not alter solubility [20] and, importantly, the pattern of ~- phosphorylation in AD is similar to normal development where PHF are not noted [3,13,23,28].

Based on the insolubility characteristics and the relative longevity of the pathological lesions in Alzheimer disease, we suggested that the protein components of the neurofibrillary tangle proteins are composed of crosslinked proteins that are resistant to limited proteolysis [5,55,59]. Our data supports this concept, since all solubilized protein were represented as smears on SDS-PAGE. Size heterogeneity (smears) of high to low molecular mass may reflect released proteins that have been attacked by proteases in vivo and only maintained in the PHF structure due to crosslinks (Fig. 7). Further, AGE modifications, including pyrraline and pentosidine, have been demonstrated in NFT and soluble PHF [30,58,67]. Here we directly demonstrate further that in vitro glycation of soluble PHF transforms it into 'insoluble PHF' with analogous solubility properties to insoluble PHF isolated from neurofibrillary tangles. Consistent with this, we showed insoluble PHF appear to be extensively crosslinked; possibly related to extensive AGE modification of ~- and possibly other protein components [30,55,58,59,63,67]. In contrast, we found senile plaques which contain AGE [58] are somewhat soluble in denaturants such as formic acid (Smith et al., unpublished observations), indicating the protein components of senile plaques are not as extensively crosslinked as those in PHF.

Glycation and oxidative stress are synergistic parallel processes [25,59] and as such, one can confidently predict that a number of glycoxidative modifications contribute towards the pathogenesis of AD [59]. In this regard, it has already been demonstrated that the neurofibrillary pathol-

A

B

C

D

PHF

Intact Subunits

Partially Proteolyzed ~ ~ ' x J ~f~ Subunits

~ S°lubilized | $ ~ ' f ' "

Cmss-lizlked Subunits

PAGE

Fig. 7. Four possible models of insoluble PHF with different contribution of proteolysis and crosslinking. PHF on the left are shown in cross section with 8 subunits. The subunits are partially proteolyzed in B and D or crosslinked in C and D. To the right are the expected electrophoretic pattern on SDS-PAGE. The observed data (Fig. 2) are most like D, i.e., crosslinked PHF subunit proteins which have been subjected to proteolysis either from the experimental procedures, postmortem delay or from in vivo attempts of proteolytic removal.

ogy of AD contains carbonyl-modified neurofilaments [57]; lipid peroxidation products such as malondialdehyde [67], and oxidized cysteine residues [50].

The presence of AGE, posttranslational modifications initiated by a condensation reaction between protein amino groups and reducing sugars via the Maillard reaction suggests further that oxidative stress is a major modulator in the pathogenesis of AD. Indeed, the synergistic nature of glycation and oxidation is shown by the fact that glycated proteins invariably contain oxidative modifications [25]. Therefore, in Alzheimer disease, glycation and oxidation would lead to the formation of crosslinking intermediates that would alter the biochemical and physical properties of reactive proteins [59]. Supporting an important role for oxidative stress in Alzheimer disease pathogenesis are several independent pieces of evidence. First, several an- tioxidant enzymes are markedly increased including heme oxygenase-1 [54] and superoxide dismutase [9,41]. Second, PHF are associated with AGE modifications including pyrraline and pentosidine [58] and soluble PHF is modified by AGE [30,67]. Third, the occurrence of ubiquitin [43]


and other heat shock proteins, such as HSP27 [46], HSP70

[21] and a B - c r y s t a l l i n [33] within neurofibr i l lary tangles

is also an indicat ion o f oxida t ive stress. Fourth, oxidat ion

o f soluble amyloid-/3, or amyloid-/3 conta ining fragments ,

causes crossl inking and aggregat ion [7]. Fifth, the forma-

tion o f SDS-res is tant complexes be tween apol ipoprote in E

and amyloid-/3 is increased under oxid iz ing condi t ions and

can be comple te ly prevented under reducing condi t ions

[60]. Sixth, iron, a free radical catalyst, is e levated in

neurofibr i l lary tangle-bear ing neurons [16].

The apparent impor tance of glycat ion in the pathogene-

sis of A l z h e i m e r disease might lead one to expec t a

correlat ion with diabetes mell i tus. There are several caveats

that might c o m p o u n d these invest igat ions as recent ly dis-

cussed [56]. First, several sugars and other compounds

(e.g. ascorbate), in addit ion to glucose, more readily fo rm

advanced glycat ion end products than glucose; second,

diabetes might lead to protec t ive compensa tory changes

p romot ing an inverse relat ionship be tween A l z h e i m e r dis-

ease and diabetes; and finally, the decreased life ex-

pectancy of diabetics might reduce the inc idence o f an

age-re la ted disease. All of these aspects must be careful ly

assessed by ep idemio log ica l studies.

In conclus ion, our data s trongly supports the idea that

oxidat ive stress and the format ion o f g lyca t ion-dependent

protein crossl inks are important in A l z h e i m e r disease.

Therefore , we suggest that strategies based on amel iora t ing

oxida t ive damage might provide a therapeutic inroad to

increase in v ivo remova l o f the lesion and poss ibly s low

the progress ion of A l z h e i m e r disease.

Acknowledgements

W e thank Drs. Stanley Prusiner, Sharon Greenberg and

Te t sufumi U e d a for valuable advice and encouragement ,

Drs. Luci l ia Aut i l io -Gambet t i , Les ter Binder, Peter Davies ,

Sharon Greenberg , Khal id Iqbal, Anne Johnson and Ken-

neth S. Kosik for p rov id ing immuno log ica l reagents, and

Sandra B o w e n for manuscr ipt preparation.

This work was supported by Nat ional Insti tutes of

Heal th grants AG09287 , AG07552 and AG08992 . M A S is

a Dal land Fe l low of the Amer ican Phi losophica l Society.

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Quantitative solubilization and analysis of insoluble paired helical filaments from alzheimer disease