©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Dephosphorylation of Alzheimer Paired Helical Filaments by Protein Phosphatase-2A and -2B (*)

(Received for publication, September 15, 1994; and in revised form, December 6, 1994)

Jian-Zhi Wang Cheng-Xin Gong Tanweer Zaidi Inge Grundke-Iqbal Khalid Iqbal (§)

From the New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Microtubule-associated protein is abnormally hyperphosphorylated in the brain of patients with Alzheimer disease and in this form is the major protein subunit of the paired helical filaments (PHF), the most prominent lesion of the disease. In this study the dephosphorylation of sparingly soluble PHF, PHF II- by brain protein phosphatase (PP)-2A(1) and PP-2B, and the resulting biochemical, biological, and structural alterations were investigated. Both of the phosphatases dephosphorylated PHF II- at the sites of Ser-199/Ser-202 and partially dephosphorylated it at Ser-396/Ser-404; in addition, PHF II- was dephosphorylated at Ser-46 by PP-2A(1) and Ser-235 by PP-2B. The relative electrophoretic mobility of PHF II- increased after dephosphorylation by either enzyme. Divalent cations, manganese, and magnesium increased the activities of PP-2A(1) and PP-2B toward PHF II-. Dephosphorylation both by PP-2B and PP-2A(1) decreased the resistance of PHF II- to proteolysis by the brain calcium-activated neutral proteases (CANP). The ability of PHF II- to promote the in vitro microtubule assembly was restored after dephosphorylation by PP-2A(1) and PP-2B. Microtubules assembled by the dephosphorylated PHF II- were structurally identical to those assembled by bovine used as a control. The dephosphorylation both by PP-2A(1) and PP-2B caused dissociation of the tangles and the PHF; some of the PHF dissociated into straight protofilaments/subfilaments. Approximately 25% of the total was released from PHF on dephosphorylation by PP-2A(1). These observations demonstrate that PHF II- is accessible to dephosphorylation by PP-2A(1) and PP-2B, and dephosphorylation makes PHF dissociate, accessible to proteolysis by CANP, and biologically active in promoting the assembly of tubulin into microtubules.


INTRODUCTION

Microtubules are required for the integrity of the neuronal cytoskeleton and the axonal transport. Alzheimer disease (AD) (^1)is characterized by replacement and displacement of microtubules by paired helical filaments (PHF) in certain selected brain neurons, especially in hippocampus. PHF exist as neurofibrillary tangles in neuronal cell bodies, as neuropil threads in the dystrophic neurites of affected neurons, and in degenerating neurites surrounding the extracellular amyloid in neuritic (senile) plaques. Microtubule-associated protein is the major protein subunit of PHF (Grundke-Iqbal et al., 1986a; Iqbal et al., 1989; Lee et al., 1991). in AD brain, especially PHF, is abnormally phosphorylated (Grundke-Iqbal et al., 1986b; Iqbal et al., 1986, 1989; Lee et al., 1991). The abnormally phosphorylated from AD brain contains 5-9 mol of phosphate/mol of the protein, which is about three to four times the level of phosphate in normal brain (Köpke et al., 1993). PHF- is phosphorylated at multiple sites (Iqbal et al., 1989). So far 21 abnormal phosphorylation sites of PHF- (all Ser/Thr sites) have been identified either by phosphorylation-dependent antibodies (Grundke-Iqbal et al., 1986b; Iqbal et al., 1989; Brion et al., 1991; Lee et al., 1991; Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Kanemaru et al., 1992) or by mass spectrometry (Hasegawa et al., 1992; Morishima-Kawashima et al., 1995). Most of the phosphates are clustered at two sites, one amino-terminal (Ser-198 to Thr-217) and one carboxyl-terminal (Ser-396 to Ser-422) to the microtubule-binding repeat domains (Gln-244 to Gly-367); numbering according to the largest isoform, (Goedert et al., 1989). As a result of the abnormal hyperphosphorylation, the apparent molecular weight of PHF- on SDS-polyacrylamide gels is higher than that of normal (Grundke-Iqbal et al., 1986b; Iqbal et al., 1989; Lee et al., 1991). Furthermore, PHF- does not promote the assembly of tubulin into microtubules unless it is dephosphorylated prior to its interaction with tubulin (Iqbal et al., 1994).

from AD brain can be biochemically isolated into three populations (Köpke et al., 1993): (i) non-abnormally phosphorylated cytosolic (C-), (ii) soluble abnormally phosphorylated (AD P-) and, (iii) abnormally phosphorylated polymerized into PHF (PHF-). Based on the solubility, PHF- can be further classified into two species: PHF I- and PHF II-. PHF I- is readily soluble in 2.0% SDS, whereas PHF II- requires ultrasonication and heating in SDS for extractions (Iqbal et al., 1984). Pathologically, these species of may reflect different stages of neuronal degeneration in AD brain, that is, normal (C-) first becomes abnormally hyperphosphorylated (AD P-) and then by a presently unknown mechanism becomes polymerized into PHF (PHF-); unlike AD P- and PHF I-, PHF II- also becomes partly ubiquitinated (Grundke-Iqbal et al., 1988; Morishima-Kawashima et al., 1993).

Phosphoseryl and phosphothreonyl protein phosphatases, which are classified into four major types, i.e. PP-1, PP-2A, PP-2B, and PP-2C (for review, see Cohen(1989)), are present in significant amounts in human brain (Gong et al., 1993). We have previously found that PP-2C is not active toward any abnormal sites of AD P-, whereas PP-1 is only effective toward two of the epitopes investigated (Gong et al., 1994b). Therefore, PP-2A and PP-2B are the most probable candidate phosphatases involved in the abnormal phosphorylation of in AD and for this reason were chosen for this study.

Previous studies have shown that AD P- can be dephosphorylated by PP-2A(1) (equally well by PP-2A(1) and PP-2A(2)) and PP-2B at several abnormal phosphorylation sites (Gong et al., 1994a and 1994c), implying that the neurodegenerative changes in AD in their early stage might be reversible.

In the present study, dephosphorylation of PHF II-, a late stage of Alzheimer neurofibrillary pathology (Bancher et al., 1989; Köpke et al., 1993) by brain PP-2A(1) and PP-2B and the resulting biochemical, biological, and structural alterations were investigated. PHF II- were found to be partially accessible to dephosphorylation by PP-2A(1) and PP-2B, and treatment with these phosphatases caused dissociation of PHF/tangles, decreased the protease resistance, and restored the microtubule assembly-promoting activity of PHF II-.


MATERIALS AND METHODS

Human brains employed for this study were obtained within 6 h postmortem and stored frozen at -75 °C until used. Protein phosphatase 2A(1) and 2B were purified from bovine brain according to the methods described by Cohen et al.(1988) and Sharma et al.(1983), respectively. CANP (a mixture of micro- and millimolar calcium-dependent enzymes) was purified from calf brain by the procedure described previously (Malik et al., 1983). Polyclonal antibody 92e and 102c were raised as reported previously (Grundke-Iqbal et al., 1988; Iqbal et al., 1989). Monoclonal antibodies -1 and PHF-1 were provided by Drs. L. I. Binder (Binder et al., 1985) and S. Greenberg (Greenberg et al., 1992), respectively. SMI31, SMI33, and SMI34 were purchased from Sternberger Monoclonals Inc., Baltimore, MD. Alkaline phosphatase-conjugated anti-mouse and anti-rabbit IgG were purchased from Sigma. I-Labeled donkey anti-rabbit whole antibody was from Amersham (Arlington, IL).

Isolation of PHF II

PHF II were isolated from histopathologically confirmed AD brains according to the long procedure of Iqbal et al.(1984). Briefly, cerebral cortex, cleaned free of white matter and meninges, was fractionated by a combination of sieving through nylon bolting cloth, treatment with 2% sodium dodecyl sulfate (SDS), and by discontinuous sucrose density gradients and glass beads chromatography.

Isolation of AD P- and Normal Human

AD P- was isolated from the 27,000 times g to 200,000 times g fraction of the Alzheimer brain homogenate by extraction in 8 M urea, followed by acid precipitation and dialysis against Mes buffer as described previously (Köpke et al., 1993). This AD P--enriched fraction is readily soluble in SDS-free buffer.

Normal human was purified as described previously (Köpke et al., 1993) from 35-45% ammonium sulfate precipitates of the 200,000 times g brain supernatant, followed by acid treatment (pH 2.7) and chromatography on a phosphocellulose column (Cellulose phosphate P11, Whatman).

Dephosphorylation Assay

Unless otherwise specified, the dephosphorylation of PHF II- was carried out at 37 °C for 45 min in 50 mM Tris-HCl (pH 7.0), 20 mM beta-mercaptoethanol, 0.1 mg/ml of bovine serum albumin, 1.0 mM MnCl(2), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 70 µg/ml PHF II- either with 2.0 units/ml PP-2A(1) or 5.0 units/ml PP-2B (one unit of PP-2A(1) or PP-2B is defined as the amount of the enzyme that releases 1.0 nmol of phosphate/min from [P]phosphorylase a or [P]phosphorylase kinase at 30 °C, respectively). The reaction mixture for dephosphorylation by PP-2B also included 1.0 mM CaCl(2) and 1 µM calmodulin. Dephosphorylation of AD P- was carried out under identical conditions as that of PHF II-, except with lower concentrations of the phosphatases (see Fig. 2). The reaction was initiated by the addition of enzyme and terminated by precipitation with 4 volumes of cold acetone. Dephosphorylation was detected by Western blots as described below.


Figure 2: Dephosphorylation of PHF II- and AD P- by various concentrations of PP-2A(1) and PP-2B. Western blots of PHF II- (a, 2.0 µg/lane) and AD P- (b, 0.5 µg/lane) were carried out using -1 as primary antibody after incubation either without (a and b, lane 1) or with PP-2B (a, lanes 2-4; b, lanes 2-5) or with PP-2A(1) (a, lanes 5-7; b, lanes 6-9) at 37 °C for 45 min. In a, the concentrations of PP-2B for lanes 2-4 were 1.0 units/ml, 2.5 units/ml, and 5.0 units/ml, respectively, whereas those of PP-2A(1) for lanes 5-7 were 0.5 unit/ml, 1.0 unit/ml, and 2.0 units/ml, respectively. The molecular mass (kDa) standards are shown at the left of a. In b, PP-2B for lanes 2-5 was used at concentrations of 0.36 unit/ml, 0.91 unit/ml, 1.82 units/ml, and 3.63 units/ml, whereas PP-2A(1) for lanes 6-9 was 0.1 unit/ml, 0.25 unit/ml, 0.5 unit/ml, and 1.0 unit/ml, respectively. A higher concentration of the phosphatases was required to dephosphorylate PHF II- than AD P-.



Western Blots

The precipitated protein samples were dissolved in SDS-PAGE sample buffer and heated in boiling water for 5 min, followed by 5-15% gradient SDS-PAGE as described by Laemmli (1970). Immunoblotting was carried out as described previously (Grundke-Iqbal et al., 1984, 1986b). The dilution of the primary antibodies used and their epitopes are shown in Table 1. The blots were developed by alkaline phosphatase-conjugated secondary antibodies and 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt and p-nitro blue tetrazolium chloride as substrates.



Quantitation of Release of Total Protein and from PHF

PHFs, untreated or treated with 2 units/ml PP-2A(1) for 3 h at 37 °C, as described above, in the presence or absence of 10 µM okadaic acid, were centrifuged for 15 min at 16,000 times g. The pellet was washed once as above. The total protein in the pellet and the two combined supernatants was measured by the method of Bensadoun and Weinstein(1976). The amount of in these fractions was determined by the radioimmuno-dot blot assay of Khatoon et al.(1992). Polyclonal anti- antibody 92e (1:2500) and I-labeled donkey anti-rabbit whole antibody (0.1 µg/ml) were employed as primary and secondary antibodies, respectively, for developing the blots.

Proteolysis by Calcium-activated Neutral Proteases

The effect of dephosphorylation on the CANP proteolysis of PHF II- was studied as follows. PHF II- were dephosphorylated with either PP-2A(1) or PP-2B as described above, except that protease inhibitor leupeptin was excluded. The dephosphorylation reaction was terminated by boiling the assay mixture for 5 min and the proteolysis reaction was initiated by adding a final concentration of 0.5 mM CaCl(2) and 0.25 unit/ml CANP. One unit of CANP activity is defined as the amount of the enzyme which catalyzed an increase of 1.0 absorbance unit at 280 nm in 1 h at 37 °C by using dimethylated casein (Sigma, C9801) as a substrate (Kawashima et al., 1984). Aliquots were removed at 0, 5, 15, 30, 60, 90, and 180 min, and the reaction was terminated by the addition of 4 volumes of cold acetone. Aliquots from each sample containing 2.0 µg of PHF II- protein were electrophoresed on 5-15% SDS-PAGE and immunoblotted with polyclonal antibody 92e.

Extraction of PHF II- and in Vitro Assembly of Microtubules

PHF II-, untreated or treated with PP-2A(1) or PP-2B as described above, were washed first with phosphate buffer and then with assembly buffer followed by ultrasonication and extraction of as described previously (Iqbal et al., 1994). Rat brain tubulin was isolated through two temperature-dependent cycles of microtubule polymerization-depolymerization (Shelanski et al., 1973) followed by phosphocellulose ion-exchange column chromatography (Sloboda and Rosenbaum, 1979). The in vitro microtubule assembly was carried out by incubating, at 37 °C in 1-cm quartz microcuvettes, rat tubulin (3.0 mg/ml) with extracted (0.2 mg/ml) in an assembly buffer containing 100 mM Mes, 1 mM EGTA, and 2 mM GTP. The assembly was followed up to 15 min by recording the turbidimetric changes at 350 nm in a Cary 1 spectrophotometer. At the end of the assembly reaction, aliquots of the incubation mixture were examined by negative stain electron microscopy as described previously for microtubules (Wisniewski et al., 1984; Iqbal et al., 1986).

Negative Stain Electron Microscopy of PHF

The effect of dephosphorylation on the structure of PHF II was examined by negative stain electron microscopy as described previously (Wisniewski et al., 1984; Köpke et al., 1993). Each experiment on the effect of dephosphorylation on the structure of PHF was carried out at least two times.


RESULTS

Both PP-2A(1) and PP-2B Dephosphorylate PHF II- at Several Sites

Using a panel of phosphorylation-dependent, site-specific antibodies (Table 1), we found that both PP-2A(1) (2.0 units/ml) and PP-2B (5.0 units/ml) unblocked antibody -1 epitope (Fig. 1a), suggesting that the enzymes dephosphorylated PHF II- at Ser-199/Ser-202. In addition, PP-2A(1) and PP-2B dephosphorylated, respectively, Ser-46 (Fig. 1b) and Ser-235 (Fig. 1c) as determined by immunostaining with antibodies 102c and SMI33. Both of the enzymes partially masked SMI31 (Fig. 1d) and SMI34 (Fig. 1e) epitopes, indicating that they partially dephosphorylated Ser-396/Ser-404 and changed the conformation of in PHF II. The dephosphorylation of PHF II- at Ser-396/Ser-404 by either enzyme was less obvious with PHF-1 than that seen with SMI31 antibody (Fig. 1f). Immunoblot with polyclonal antibody 92e showed an electrophoretic mobility shift in after dephosphorylation by PP-2A(1) or PP-2B (Fig. 1g). These results revealed that in PHF II can be selectively dephosphorylated in vitro by PP-2A(1) and PP-2B.


Figure 1: Dephosphorylation of PHF II- by PP-2A(1) and PP-2B. Western blots of PHF II- with different antibodies were carried out after incubation either without (lane 1) or with 5.0 units/ml PP-2B (lane 2) or with 2.0 units/ml PP-2A(1) (lane 3) at 37 °C for 45 min as described under ``Materials and Methods.'' Lane 4 is untreated normal human as a control. Six phosphorylation-dependent, site-specific monoclonal antibodies and one polyclonal anti- antibody were employed for immunoblotting as shown beneath each panel. The epitope of each antibody is described in Table 1. Molecular mass (kDa) standards are indicated at the left of a. The amounts of protein/lane were 2 µg for 92e and -1, 3 µg for PHF-1 and 102c, and 5 µg for SMI31, SMI33, and SMI34, respectively. Both PP-2A(1) and PP-2B dephosphorylated Ser-199/Ser-202 (-1) and partially dephosphorylated Ser-396/Ser-404 (SMI31 and PHF-1). PP-2A(1) and PP-2B also dephosphorylated Ser-46 (102c) and Ser-235 (SMI31), respectively. In addition, dephosphorylation by either phosphatases changed the conformation (SMI34) and shifted the mobility (92e) of in PHF II. The prominent immunostaining of the PHF high molecular mass smear by antibody 102e (Ser 46) in b, lane 3, suggests that the smear selectively contains isoforms with the amino-terminal insert(s). Polypeptides with a mass of less than 43 kDa represent the degraded .



Higher Concentration of the Phosphatases Is Required to Dephosphorylate in PHF II than AD P-

Dephosphorylation by PP-2B was unable to unmask -1 epitope of PHF II- at a concentration of 1.0 unit/ml, and dephosphorylation by PP-2A(1) at 0.5 unit/ml resulted in a weak immunostaining. The maximal labeling of PHF II- with -1 was obtained at a concentration of either 2.0 units/ml PP-2A(1) or 5.0 units/ml PP-2B (Fig. 2a). On the other hand, PP-2A(1) and PP-2B readily dephosphorylated AD P- at -1 site at the concentration of 0.1 unit/ml and 0.36 unit/ml, respectively, although the extent of the dephosphorylation was increased with the increase of the enzyme concentration up to 1.0 unit/ml PP-2A(1) and 3.6 units/ml PP-2B (Fig. 2b). No inhibitory activity toward the phosphatases was found in PHF II- sample (data not shown). These results indicated that compared with AD P-, PHF II- is a less favorable substrate for dephosphorylation by PP-2A(1) and PP-2B. Additionally, PP-2A(1) is more active than PP-2B toward PHF II-.

The Phosphatase Activities toward PHF II- Are Affected by Divalent Cations and Other Effectors

Manganese activated the dephosphorylation of PHF II- by PP-2A(1) or PP-2B (Fig. 3, a and b, compare lanes 2, 3, and 4 with lane 11). The activity increased with the increase of manganese concentration and appeared to reach the maximal at the concentration of 0.1 mM, both in the case of PP-2A(1) and PP-2B. Magnesium stimulated the activity of both enzymes at concentrations of 1.0 mM and 10.0 mM but not at 0.1 mM (Fig. 3, a and b, compare lanes 5, 6, and 7 with lane 11). Calcium and calmodulin activated PP-2B (Fig. 3b, compare lane 8 with lane 11). However, higher activity was obtained when manganese or magnesium were also included (Fig. 3b, compare lanes 9 and 10 with lane 8 and with lane 11). PP-2A(1) activity was minimally affected by polylysine (Fig. 3a, compare lanes 8, 9, and 10 with lane 11). The data suggested that Mn and Mg stimulated PP-2A(1) and PP-2B, Ca/calmodulin-activated PP-2B, whereas polylysine minimally promoted PP-2A(1) activity.


Figure 3: Effects of divalent cations and other effectors on the dephosphorylation of PHF II- by PP-2A(1) (a) and PP-2B (b). Western blots of PHF II- (2 µg/lane) with antibody -1 were carried out after incubation of the substrate either without phosphatase (a and b, lane 1) or with phosphatase (PP-2A(1), 2 units/ml; PP-2B, 5 units/ml) and different effectors (a and b, lanes 2-11). The molecular mass (kDa) markers are shown at the left of the panels. In a and b, the reaction mixture for lanes 2-4 contained 0.01 mM, 0.1 mM, or 2.0 mM MnCl(2) and for lanes 5-7 contained 0.1 mM, 1.0 mM, or 10.0 mM MgCl(2). Lanes 8-10 in a were with 1.0, 10.0, and 100.0 µM polylysine, whereas lanes 8-10 in b were with 1.0 mM CaCl(2), 1.0 µM calmodulin; 1.0 mM CaCl(2), 1.0 µM calmodulin, 1.0 mM MnCl(2); and 1.0 mM CaCl(2), 1.0 µM calmodulin, 1.0 mM MgCl(2), respectively. Lane 11 was with 5.0 mM EDTA in a and 5.0 mM EGTA in b, respectively. Mn and Mg activated both phosphatases, whereas Ca/calmodulin stimulated PP-2B. Polylysine minimally promoted the PP-2A(1) activity.



Dephosphorylation of PHF II- by PP-2A(1) and PP-2B Increases Its Proteolysis by CANP

PHF II- could not be digested by CANP in 3 h (Fig. 4a). In contrast, PHF II- dephosphorylated by PP-2A(1) (Fig. 4b) or PP-2B (Fig. 4c) showed significant proteolysis after incubation with CANP for 3 h. These results suggested that PHF II- is resistant to proteolysis by CANP, and prior dephosphorylation of PHF II- by PP-2A(1) or PP-2B can decrease this resistance considerably.


Figure 4: Dephosphorylation-induced CANP proteolysis of PHF II-. PHF II- (2 µg/lane) was incubated with 0.25 unit/ml CANP for different times (as indicated beneath each lane) untreated (a) or dephosphorylated by PP-2A(1) (b) or by PP-2B (c) as described under ``Materials and Methods.'' After incubation with CANP, samples were subjected to Western blotting with polyclonal antibody 92e. Dephosphorylation of PHF II- by either enzymes decreased its resistance to CANP proteolysis. The molecular mass (kDa) markers are shown at the left of the panels.



Dephosphorylation of PHF II- by either PP-2A(1) or PP-2B Restores Its in Vitro Microtubule Assembly-promoting Activity

Incubation at 37 °C of nondephosphorylated PHF II- (PHF control) with rat tubulin in the assembly buffer generated a lower turbidimetric reading than tubulin control (Fig. 5), and no microtubules were seen by electron microscopy (Fig. 6b). In contrast, incubation of rat tubulin with PHF II- dephosphorylated by either PP-2A(1) or PP-2B generated a markedly increased turbidity at 350 nm (Fig. 5), and large numbers of microtubules were seen by negative stain electron microscopy (Fig. 6, c and d). No ultrastructural difference could be observed between the microtubules assembled by bovine used as a control (Fig. 6a) and those by dephosphorylated PHF II- (Fig. 6, c and d).


Figure 5: Effect of dephosphorylation of PHF II- by PP-2A(1) and PP-2B on its microtubule assembly-promoting activity. PHF II- was dephosphorylated and then extracted as described under ``Materials and Methods.'' Microtubule assembly was carried out by incubating at 37 °C rat brain tubulin (3.0 mg/ml) with bovine or PHF II- (0.2 mg/ml). Microtubule assembly-promoting activity was increased after phosphatases treatment. Curves show microtubule assembly in the presence of normal , PHF dephosphorylated with PP-2A(1) and PP-2B, PHF untreated with any phosphatase, and assembly with tubulin alone.




Figure 6: Electron micrographs showing the products of microtubule assembly negatively stained with phosphotungstic acid. Aliquots of each sample (from Fig. 5) were taken at steady state of polymerization and stained negatively with 2% phosphotungstic acid as described under ``Materials and Methods.'' Large numbers of microtubules from tubulin were observed in the presence of bovine and PP-2A(1)- and PP-2B-treated PHF (a, c, and d, respectively); no microtubules were seen in the presence of nondephosphorylated PHF II- (b), and an occasional microtubule could be observed in the tubulin alone control (not shown). No ultrastructural differences were observed among microtubules assembled with the bovine control and the phosphatases-treated PHF II-. Bar, 0.5 µm.



Dephosphorylation of PHF II- Leads to Their Dissociation

The negative stain electron microscopy revealed a dissociation of the neurofibrillary tangles after incubation of PHF II- with PP-2A(1) (Fig. 7b) or PP-2B (Fig. 7c) at 37 °C for 45 min. The PHF tangles dissociated into individual PHF, and the twists in PHF started becoming less defined. Further dissociation of the PHF and the tangles was seen by 3-h dephosphorylation either by PP-2A(1) (Fig. 7e) or by PP-2B (Fig. 7f). An increasing dissociation of the tangles into individual PHF and dissociation of PHF into straight subfilaments/protofilaments was frequently seen in the 3-h dephosphorylated samples, but not in the no phosphatase added control samples (Fig. 7d). Quantitation by radioimmuno-dot blots revealed a net release of approximately 25% of total from PHF following dephosphorylation by PP-2A(1) (Table 2).


Figure 7: Electron micrographs of PHF II- before and after phosphatases treatment. Incubation of PHF II- was carried out at 37 °C for 45 min (a, b (two panels), and c (three panels)) or 3 h (d, e, and f) either in the absence (a and d) or the presence of PP-2A(1) (b and e) or PP-2B (c and f). The dephosphorylation was terminated by the addition of 100 mM of phosphate buffer (pH 7.5), and the grids were prepared as described under ``Materials and Methods.'' Marked structural changes could readily be seen after 3-h dephosphorylation by either enzyme. Bars, 0.1 µm.






DISCUSSION

One of the most characteristic brain lesions of AD is the formation of PHF in the affected neurons. The abnormal phosphorylation of probably precedes its polymerization into PHF (Bancher et al., 1989; Köpke et al., 1993). Previously, we have demonstrated that this pre-PHF abnormal , AD P-, can be dephosphorylated at several abnormal phosphorylation sites by PP-2A(1) and PP-2B (Gong et al., 1994c, 1994a) and that both of these enzymes are localized in neurons, including neurons predilected for neurofibrillary tangles (Pei et al., 1994). Furthermore, Drewes et al.(1993) have reported that the extracted from PHF by detergents and made soluble can be dephosphorylated at the AT8 site (this antibody recognizes Ser-199/Ser-202 when phosphorylated) by PP-2A and PP-2B. In the present study, we have investigated whether polymerized into PHF/neurofibrillary tangles is also accessible to protein phosphatases and have examined the effect of dephosphorylation on the structure, proteolysis, and the biological activity of in PHF. We found (i) that PHF II- can be dephosphorylated at Ser-46 by PP-2A(1), at Ser-235 by PP-2B, and at Ser-199/Ser-202 and partially at Ser-396/Ser-404 by both PP-2A(1) and PP-2B; (ii) that the dephosphorylation of PHF II- by either phosphatase decreases its resistance to proteolysis by CANP; (iii) that the dephosphorylation of PHF II- with either phosphatase restores its biological activity as determined by the microtubule assembly-promoting activity; and (iv) that the dephosphorylation leads to a dissociation of the neurofibrillary tangles and individual PHF into straight protofilaments and release of . These data suggest that not only soluble abnormally phosphorylated but also the polymerized into PHF is accessible, although to a lesser degree, to the protein phosphatases PP-2A(1) and PP-2B, and on dephosphorylation with these enzymes the structure of PHF dissociates and becomes biologically active and can be proteolyzed by CANP.

Compared with AD P-, PHF II- is less amenable to dephosphorylation by PP-2A(1) and PP-2B. The phosphates at -1 epitope in AD P- were readily removed by 0.1 unit/ml PP-2A(1) or 0.36 unit/ml PP-2B, whereas for PHF II-, 2.0 units/ml PP-2A(1) or 5.0 units/ml PP-2B were required for maximal dephosphorylation at the same epitope. Pathologically, AD P- and PHF II- may reflect, respectively, an early stage and a late stage of AD neurofibrillary degeneration. The structural state of in PHF might be responsible for its decreased accessibility to the phosphatases. The same reason may also explain why AD P- can be dephosphorylated at the abnormal sites Ser-396/Ser-404 by PP-2A and PP-2B (Gong et al., 1994a, 1994c), whereas PHF II- can be only partially dephosphorylated at these sites. The exact reason for the relative resistance of Ser-396/Ser-404 sites for dephosphorylation in PHF II- by PP-2A(1), and PP-2B is at present not known. It is likely that the carboxyl-terminal region of the abnormally phosphorylated is buried into the PHF polymer and is less accessible to the phosphatases.

The effect of divalent cations on the phosphatase activities showed that Mn stimulated PP-2A(1) and PP-2B at the concentration of 0.01 mM, whereas 1.0 mM Mg was required for comparable activities. These concentrations of Mn and Mg are in the physiological range (Friberg et al., 1986). The stimulation of the phosphatase activities by these metals might have physiological and therapeutic significance.

Using soluble AD P- as a substrate and rat brain phosphatase as an enzyme source, we (Gong et al., 1994a, 1994c) demonstrated previously that in vitro PP-2B was more active than PP-2A. Similar results were reported by Drewes et al. (1993). In the present study, we found that bovine brain PP-2A(1) was more active than PP-2B toward PHF II-, suggesting that both PP-2A(1) and PP-2B might play a role in the abnormal phosphorylation of and that the differences between the previous and the present studies might be related to the use of different substrates and possibly different enzyme sources.

Although the mechanism for PHF formation and accumulation as tangles remains largely unknown, one can postulate that an impaired proteolysis of abnormally phosphorylated might be related to this pathological processing. Previous studies have shown that PHF is extremely protease-resistant (Grundke-Iqbal et al., 1988; Wischik et al., 1988). In the present study we have found that dephosphorylation of PHF II- by PP-2A(1) and PP-2B increases its proteolysis by CANP. These findings suggest that abnormal phosphorylation of PHF- might be involved in its increased resistance to CANP digestion, and this abnormal phosphorylation-induced inhibition of proteolysis by CANP can be reversed by the dephosphorylation of the abnormal by PP-2A(1) and PP-2B. We also found that dephosphorylated AD P- was degraded significantly more rapidly and extensively than PHF II- (data not shown). These differences in the CANP proteolysis of AD P- and PHF II- might be related to the nonpolymeric state and a more complete dephosphorylation of the former than the latter.

Interestingly, it was also found that normal human was rapidly digested by the CANP used in this study but not by that purchased from Sigma (data not shown). Johnson et al.(1989) also reported that purified from total brain heat-stable fraction was resistant to degradation by the CANP from Sigma. The CANP from Sigma was purified from rabbit skeletal muscle and only contained the millimolar type of CANP, whereas the CANP used in the present study was purified from calf brain and contained both millimolar and micromolar types of CANP. Thus, the difference in the activities of CANP toward between the Sigma enzyme and the enzyme used in the present study might be either due to the tissue specificity or the type specificity or both; tissue-specific CANP have been reported recently (Sorimachi et al., 1994).

Previous studies have shown that soluble abnormally phosphorylated and PHF II- isolated from AD brain had no microtubule assembly activity. However, when this was extensively dephosphorylated by alkaline phosphatase, the microtubule assembly-promoting activity was restored (Alonso et al., 1994; Iqbal et al., 1994). In the present study, we found that partial dephosphorylation of PHF II- by protein phosphatase 2A(1) or 2B also restored its biological activity in promoting the in vitro microtubule assembly. The assembly activity of the PP-2A(1)-treated sample as determined by the turbidimetric changes was much higher than that of the PP-2B-treated sample. These findings suggest (i) that the phosphorylation of at Ser-235, a site dephosphorylated by PP-2B, and not by PP-2A(1), might not be involved in the microtubule assembly-promoting activity; and (ii) that one or more selective sites that are dephosphorylated by PP-2A(1) but not by PP-2B might enhance the microtubule assembly activity. The nature of these differences, which will be investigated in a separate study, is, at present, not known. Although the turbidity is significantly higher in the PP-2A(1)-treated sample than PP-2B-treated sample, no obvious difference in the structure of microtubules was seen by the negative stain electron microscopy.

PHF are ultrastructurally distinct from elements of normal neuronal cytoskeleton, i.e. microtubules and neurofilaments. PHF have a diameter of 22-24 nm, which narrows to 10 nm at every 80-nm interval (Kidd, 1964; Wisniewski et al., 1984). In this study, we observed that dephosphorylation of PHF by either PP-2A(1) or PP-2B dissociated PHF tangles, and this dissociation became more significant with the increase in dephosphorylation. During the first 45 min of dephosphorylation, dissociation of PHF from the neurofibrillary tangles and a decrease in the discernability of the twists of PHF were observed. After the dephosphorylation for 3 h, a fewer number of the tangles and PHF and a concomitant release of was observed. Furthermore, an increasing number of PHF were found to lose their twist and were dissociated into straight protofilaments. Although the role of phosphorylation in the polymerization of into PHF is not understood, the present study suggests that dephosphorylation by PP-2A(1) and PP-2B might reverse this lesion.

In conclusion, the present study reveals that in PHF II is biologically inactive and protease-resistant. However, PHF in neurofibrillary tangles are accessible to dephosphorylation at some sites by PP-2A(1) and PP-2B. Dephosphorylation of PHF- restores the microtubule assembly-promoting activity, increases the proteolysis by CANP, and dissociates the neurofibrillary tangles and the PHF. Dephosphorylation might inhibit and reverse the neurofibrillary degeneration in AD brain.


FOOTNOTES

*
This work was supported in part by the New York State Office of Mental Retardation and Developmental Disabilities, National Institutes of Health Grants AG 05892, AG 08076, and NS 18105 and the Zenith Award (to K. I.) from the Alzheimer's Association, Chicago. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: NYS Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5259; Fax: 718-494-1080.

(^1)
The abbreviations used are: AD, Alzheimer disease; AD P-, abnormally phosphorylated from AD brain sedimenting at 27,000-200,000 times g, soluble and reactive with -1 antibody only after dephosphorylation; CANP, calcium-activated neutral protease(s); PHF, paired helical filaments; PHF II-, in PHF that are sparingly soluble in SDS; PP-2A or PP-2B, protein phosphatase-2A or -2B; Mes, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Toolsee J. Singh for critical reading of the manuscript; Dr. M. Malik and M. D. Fenko for the generous gift of CANP; Dr. A. Alonso and F. Connell for their help in electron microscopy; Drs. L. I. Binder and S. Greenberg for supplying antibodies -1 and PHF-1, respectively; Joanne Lopez and Padmini Reginald for secretarial assistance and the Biomedical Photography Unit of this Institute for the preparation of figures. Autopsied brain specimens from Alzheimer disease cases were provided by the Netherlands Brain Bank at the Netherlands Institute for Brain Research, Amsterdam; the Brain Tissue Resource Center (Public Health Science Grant MH/NS 31862), McLean Hospital, Belmont, MA; and the National Neurological Research Bank, Veterans Administration Medical Center, Wadsworth, Los Angeles.


REFERENCES

  1. Alonso, A. del C., Zaidi, T., Grundke-Iqbal, I., and Iqbal, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5562-5566 [Abstract]
  2. Bancher, C., Brunner, C., Lassmann, H., Budka, H., Jellinger, K., Wiche, G., Seitelberger, F., Grundke-Iqbal, I., Iqbal, K., and Wisniewski, H. M. (1989) Brain Res. 477, 90-99 [CrossRef][Medline] [Order article via Infotrieve]
  3. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241-250 [Medline] [Order article via Infotrieve]
  4. Biernat, J., Mandelkow, E.-M., Schröter, C., Lichtenberg-Kraag, B., Steiner, B., Berling, B., Meyer, H., Mercken, M., Vandermeeren, A., Goedert, M., and Mandelkow, E. (1992) EMBO J. 11, 1593-1597 [Abstract]
  5. Binder, L. I., Frankfurter, A., and Rebhun, L. I. (1985) J. Cell Biol. 101, 1371-1378 [Abstract]
  6. Brion, J. P., Hanger, D. P., Bruce, M. T., Couck, A. M., Flament-Durant, J., and Anderton, B. T. (1991) Biochem. J. 273, 127-133 [Medline] [Order article via Infotrieve]
  7. Cohen, P. (1989) Annu. Rev. Biochem. 58, 543-508
  8. Cohen, P., Alemany, S., Hemmings, B. A., Resink, T. J., Stralfors, P., and Tung, H. Y. L. (1988) Methods Enzymol. 159, 390-408 [Medline] [Order article via Infotrieve]
  9. Drewes, G., Mandelkow, E.-M., Baumann, K., Goris, J., Merlevede, W., and Mandelkow, E. (1993) FEBS Lett. 336, 425-432 [CrossRef][Medline] [Order article via Infotrieve]
  10. Friberg, L., Nordberg, G. F., and Vouk, V. B. (1986) Handbook on the Toxicology of Methods, Vol. 2, pp. 364-366, Elsevier Science Publishing Co., Inc., New York
  11. Goedert, M., Spillanti, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989) Neuron 3, 519-526 [Medline] [Order article via Infotrieve]
  12. Gong, C.-X., Singh, T. J., Grundke-Iqbal, I., and Iqbal, K. (1993) J. Neurochem. 61, 921-927 [Medline] [Order article via Infotrieve]
  13. Gong, C.-X., Singh, T. J., Grundke-Iqbal, I., and Iqbal, K. (1994a) J. Neurochem. 62, 803-806 [Medline] [Order article via Infotrieve]
  14. Gong, C.-X., Grundke-Iqbal, I., Damuni, Z., and Iqbal, K. (1994b) FEBS Lett. 341, 94-98 [CrossRef][Medline] [Order article via Infotrieve]
  15. Gong, C.-X., Grundke-Iqbal, I., and Iqbal, K. (1994c) Neuroscience 61, 765-772 [CrossRef][Medline] [Order article via Infotrieve]
  16. Greenberg, S. G., Davies, P., Schein, J. D., and Binder, L. (1992) J. Biol. Chem. 267, 564-569 [Abstract/Free Full Text]
  17. Grundke-Iqbal, I., Iqbal, K., Tung, Y.-C., and Wisniewski, H. M. (1984) Acta Neuropathol. (Berl.) 62, 259-267 [Medline] [Order article via Infotrieve]
  18. Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y.-C., Zaidi, M. S., and Wisniewski, H. M. (1986a) J. Biol. Chem. 261, 6084-6089 [Abstract/Free Full Text]
  19. Grundke-Iqbal, I., Iqbal, K., Tung, Y.-C., Quinlan, M., Wisniewski, H. M., and Binder, L. I. (1986b) Proc. Natl. Acad. Sci. U. S. A. 83, 4913-4917 [Abstract]
  20. Grundke-Iqbal, I., Vorbrodt, A. W., Iqbal, K., Tung, Y. C., Wang, G. P., and Wisniewski, H. M. (1988) Mol. Brain Res. 4, 43-52
  21. Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 267, 17047-17054 [Abstract/Free Full Text]
  22. Iqbal, K., Zaidi, T., Thompson, C. H., Merz, P. A., and Wisniewski, H. M. (1984) Acta Neuropathol. (Berl.) 62, 167-177 [Medline] [Order article via Infotrieve]
  23. Iqbal, K., Grundke-Iqbal, I., Zaidi, T., Merz, P. A., Wen, G. Y., Shaikh, S. S., Wisniewski, H. M., Alafuzoff, I., and Winblad, B. (1986) Lancet 2, 421-426 [Medline] [Order article via Infotrieve]
  24. Iqbal, K., Grundke-Iqbal, I., Smith, A. J., George, L., Tung, Y.-C., and Zaidi, T. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5646-5650 [Abstract]
  25. Iqbal, K., Zaidi, T., Bancher, C., and Grundke-Iqbal, I. (1994) FEBS Lett. 349, 104-108 [CrossRef][Medline] [Order article via Infotrieve]
  26. Johnson, G. V. W., Jope, R. S., and Binder, L. I. (1989) Biochem. Biophys. Res. Commun. 163, 1505-1511 [Medline] [Order article via Infotrieve]
  27. Kanemaru, K., Takio, K., Miura, R., Titani, K., and Ihara, Y. (1992) J. Neurochem. 58, 1667-1675 [Medline] [Order article via Infotrieve]
  28. Kawashima, S., Nomoto, M., Hayashi, M., Inomatam, M., Nakamura, M., and Imahori, K. (1984) J. Biochem. (Tokyo) 95, 95-101 [Abstract]
  29. Khatoon, S., Grundke-Iqbal, I., and Iqbal, K. (1992) J. Neurochem. 59, 750-753 [Medline] [Order article via Infotrieve]
  30. Kidd, M. (1964) Brain 87, 307-320
  31. Köpke, E., Tung, Y.-C., Shaikh, S., Alonso, A. del C., Iqbal, K., and Grundke-Iqbal, I. (1993) J. Biol. Chem. 268, 24374-24384 [Abstract/Free Full Text]
  32. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  33. Lee, V. M.-Y., Balin, B. J., Otvos, L., Jr., and Trojanowski, J. Q. (1991) Science 251, 675-678 [Medline] [Order article via Infotrieve]
  34. Lichtenberg-Kraag, B., Mandelkow, E. M., Biernat, J., Steiner, B., Schröder, C., Gustke, N., Meyer, H. H., and Mandelkow, E. (1992) Proc. Natl. Acad. Sci. U. S. A. 82, 5384-5388
  35. Malik, M. N., Fenko, M. D., Iqbal, K., and Wisniewski, H. M. (1983) J. Biol. Chem. 258, 8955-8962 [Abstract/Free Full Text]
  36. Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1993) Neuron 10, 1151-1160 [Medline] [Order article via Infotrieve]
  37. Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Yoshida, H., Titani, K., and Ihara, Y. (1995) J. Biol. Chem. 270, 823-829 [Abstract/Free Full Text]
  38. Pei, J. J., Sersen, E., Iqbal, K., and Grundke-Iqbal, I. (1994) Brain Res. 655, 70-76 [Medline] [Order article via Infotrieve]
  39. Sharma, R. K., Taylor, W. A., and Wang, J. H. (1983) Methods Enzymol. 102, 210-219 [Medline] [Order article via Infotrieve]
  40. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 765-768 [Abstract]
  41. Sloboda, R. D., and Rosenbaum, J. L. (1979) Biochemistry 18, 48-55 [Medline] [Order article via Infotrieve]
  42. Sorimachi, H., Saido, T. C., and Suzuki, K. (1994) FEBS Lett. 343, 1-5 [CrossRef][Medline] [Order article via Infotrieve]
  43. Wischik, C. M., Novak, M., Thogersen, H. C., Edwards, P. C., Runswick, M. J., Jakes, R., Walker, J. E., Milstein, C., Roth, M., and Klug, A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4506-4510 [Abstract]
  44. Wisniewski, H. M., Merz, P. A., and Iqbal, K. (1984) J. Neuropathol. Exp. Neurol. 43, 643-656 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.