microtubule-associated protein map1b showing a fetal phosphorylation pattern is present in sites of...

L , - ~ • ' ~ i l t

I ?~ i" )

_ _ (

E L S E V I E R Molecular Brain Research 26 (1994) 113-122

MOLECULAR BRAIN

RESEARCH

Research Report

Microtubule-associated protein MAP1B showing a fetal phosphorylation pattern is present in sites of neurofibrillary degeneration in brains

of Alzheimer's disease patients

Luis Ulloa a, Esteban Montejo de Garcini a, Pilar Gdmez-Ramos b, Maria A. Morfin b, Jes f i s A v i l a a,.

a Centro de Biologfa Molecular 'Severo Ochoa' (CSIC-UAM), Fac. de Ciencias, Univ. Aut6norna de Madrid, E-28049 Madrid, Spain b Departamento de Morfolog[a, Facultad de Medicina, Universidad Aut6noma de Madrid, E-28029 Madrid, Spain

Accepted 10 May 1994

Abstract

Alzheimer's disease results in the appearance of cytoskeletal disorders yielding pathological structures such a neurofibrillary tangles or dystrophic neurites. It has been previously described that the microtubule-associated protein, tau, modified by phosphorylation in serines adjacent to prolines, is a major component of these structures. Here, we show that another microtubule associated protein, MAP1B, aberrantly phosphorylated by a proline-dependent protein kinase, is a component of these previously mentioned structures. Thus, a possible common phosphorylation of axonal MAPs such as tau or MAP1B may correlate with their association with those aberrant cytoskeletal structures present in AD.

Keywords: Proline-dependent phosphorylation; Cytoskeletal disorder

1. Introduct ion

Alzheimer's disease (AD) is the most common senile dementia [25,26,51] characterized by the presence of pathological structures in brain. The two most stud- ied anatomopathological hallmarks in AD brain are neurofibrillary degeneration [30,31] and the accumulation of /3A4 amyloid protein in senile plaques [51]. Neurofibrillary degeneration is found in bundles of both straight and paired helical filaments (PHF) which accumulate in neurofibrillary tangles (NFT), neuropil threads (NT) and plaque-associated neurites. These NT, derived from axons, dendrites a n d / o r synaptic terminals [27,31], are also referred to curly fibers [6].

It has been demonstrated that PHFs are built by the microtubule-associated protein tau as a major component [16,17,18,22,26,35,64,65]. PHFs are also immuno-

* Corresponding author. Fax: (34) 1-397 4799.

0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(94)00106-0

reactive for ubiquitin [39]. NT also contain tau [27,30,31] and other cytoskeletal proteins are probably also present.

Alzheimer's disease results in neural degeneration, characterized by atrophy and loss of neurons in the neocortex, hippocampus and amygdala; the most vul- nerable cells are the pyramidal neurons [54]. However, it has been suggested that neuronal degeneration appears not to be a simple passive process, but rather an active one, since massive neurite sprouting occurs in AD brain [21,33]. This observation has promoted the hypothesis that, in AD, regeneration recapitulates development [19] in a similar manner to that of lesion-in- duced axonal sprouting, in which fetal cytoskeletal isoforms are re-expressed [8]. In this way, phosphorylated isoforms of microtubule-associated protein tau, present at early developmental stages, have been also found in AD [7,13]. Additionally, a possible fetal protein with a MW of 70 kDa is abundantly expressed in the brains of AD patients [40]. Also, another protein expressed mainly in early developmental stages, micro-

114 L. Ulloa et al. / Molecular Brain Research 26 (1994) 113-122

tubule-associated protein MAP1B, has been found in the brains of AD patients [19,53].

MAP1B is the major microtubule-associated protein found in developing neurons [4,43,45,46,48,49,60]; it is the first MAP expressed in neurons, as demonstrated by in situ localization [56]. This protein is under strong developmental regulation and it declines with progress- ing brain maturation [45]. It can be phosphorylated and two types of phospho-isoforms have been described. In one case (mode I phosphorylation), it has been suggested that the protein is modified by a proline-directed protein kinase (PDPK) [58]. The localization of these mode I-phosphorylated isoforms is restricted to developing axonal processes [12,32], and this type of modification may be related to axonal sprouting [58]. The presence of these phosphoisoforms decreases with neuron development and they are essentially not detected in the neuronal cytoplasm in adult stages, except in the olfactory bulb, where neuronal regeneration continues even in adult stages [58]. Additionally, the presence of mode I-phospho MAP1 isoforms (or related proteins) in the nuclei of neuronal cells has been suggested [45,58].

On the other hand, mode II phosphorylation appears to be the result of a modification by casein kinase II [10,57,58]. The casein kinase II-phospho- MAP1B isoforms appear to facilitate microtubule sta- bilization [10,57] and they remain present in adult stages [58].

Recent reports [19,53], suggest the presence of phosphoMAP1 isoforms in AD. Our purpose here was

to determined whether the modification of these isoforms is similar to that found in fetal stages. Our results indicate that phosphoMAP1B isoform, restricted to developing axonal processes, are reexpresed abundantly in the brains of AD patients and are local- ized at sites of neurofibrillary degeneration: neurofibrillary tangles and, mainly, at neuropil threads.

2. Materials and methods

2.1. Human brain samples

Autopsied brains from AD patients and from non-demented aged individuals were obtained from The Cambridge Brain Bank (Cam- bridge, UK), La Paz and t2 de Octubre Hospital (Madrid, Spain), Meixoeiro Hospital (Vigo, Spain) and Rush-Presbyterian-St. Luke's Medical Center (Chicago IL, USA). The neuropathological criteria used to define AD adhered to NIH age-adjusted criteria [24]. For electrophoresis, Western blotting and immuno-electron microscopy studies, unfixed blocks of tissue were frozen in dry ice within a period of 3-25 h post-mortem (Table 1). Identical conditions; amount of protein/lane and development times were used for comparative studies of AD and control samples.

2.2. Immuno-cytochemistry on tissue sections

For the immuno-cytochemical study, tissue from hippocampus, entorhinal, temporal and insular cortices was obtained (mean post- mortem time 6 h) from 11 brains: five non-demented aged (mean age 81_+6 years) and six AD cases (mean age 76+ 11 years) (Table 1). Samples were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 14-48 h, except for two blocks of tissue, one from an AD patient and the other one from a non-demented aged brain,

Table 1 Characteristics of Alzheimer's disease (AD) patients and controls

Case Cortex Sex Age Fixation Autolysis

Alzheimer FD83 TL M 79 - 22 h FDll2 TL M 74 - 24 h FD121 TL M 46 - 24 h FD124 TL F 77 - 3 h A23 TP M 82 4% para (41 h) 5 h 30 min A38 INS F 90 4% para (24 h) 5 h A42 Hpp, ENT M - 4% para (48 h) 4 h A48 TP M 70 4% para (48 h) 4 h A49 TP M 61 4% para (48 h) 14 h A57 Hpp, ENT F 81 4% formol (14 h) 4 h 30 min

Controls C521 TL F - - C522 TL F 73 - 25 h C9 INS M 73 4% para (24 h) 6 h 30 rain C15 TP F 85 4% para (47 h) 2 h C16 TP M 77 4% para (44 h) 2 h C18 TP, Hpp, ENT M 85 4% para (48 h) 8 h 30 min C22 INS M 86 4% para (20 h) l0 h 30 min

TP, temporal pole; INS, insular cortex; Hpp, hippocampal cortex; ENT, entorhinal cortex; PL, parietal lobe; TL, temporal lobe; - - , not determined. The first row with letters and numbers corresponds to the key to identify AD patients and controls.

L. Ulloa et al. / Molecular Brain Research 26 (1994) 113-122 115

which were fixed in 10% formalin for 14 h and 7 weeks, respectively (Table 1). In all cases, once cryoprotected in graded concentrations of sucrose (10-40%) in 0.1 M phosphate buffer (pH 7.4), the tissue was cut on a freezing microtome at 40 /zm and either processed immediately or kept in a glycol solution at -20°C [38,61] until use.

The primary monoclonal antibodies against anti-phosphorylated MAP1B, were 150 antibody (IgM, diluted 1:2-1:10 from a hybridome supernatant), in 3% bovine serum albumin, as previously described by Riederer et al. [47] and 125 antibody [58] (IgM, diluted 1:10 from a hybridome supernatant in 3% bovine serum albumin). Anti-tau (PHF) monoclonal antibody 423 (IgG, diluted 1 : 5) [35] and a polyclonal tau antibody (diluted 1 : 200) [37] were also used.

Immuno-cytochemistry was performed in free-floating sections, using the biotin-streptavidin-peroxidase method, with diaminobenzi- dine as a chromogen. Endogenous peroxidase was inactivated by incubating sections in a solution of 0.3% hydrogen peroxide in methanol for 30 min. Sections were incubated in 3% normal rabbit serum in phosphate-buffered saline (PBS) with 0.1% Triton X-100 at 4°C for 1 h, followed by the primary antiserum overnight at 4°C. Then sections were incubated in the corresponding biotinylated secondary antibody, either rabbit anti-mouse IgG (Biomakor, 1 : 1000) or rabbit anti-mouse IgM (Vector 1 : 1000) at room temperature for 1 h, followed by streptavidin-peroxidase (Biomakor, 1:1200) at room temperature for 1 h. To minimize the non-specific staining, pre absorption of secondary antibodies and streptavidin-peroxidase was performed with additional sections of human brain for 48 h at 4°C. To evaluate the specificity of the primary antibody staining, adjacent or closely-related tissue sections were incubated in parallel in vehicle solution without primary antibodies. Some sections were counterstained with thionin. To compare the staining obtained by the different antibodies, adjacent sections were consistently used. In order to asses the specificity of the immunocytochemical staining with the 150 antibody, selected sections from several brains were preincubated with 140/zg/ml of alkaline phosphatase (Boehringer- Mannheim, Germany) in 0,1M Tris-HCl pH 8.0, for 3 h at 32 ° with the antiprotease 'cocktail' described in Mansfield et al. [32].

2.3. Protein preparation

Microtubule protein was isolated by the procedure of Shelanski et al. [50] following the modifications of Karr et al. [23].

A MAPs preparation, enriched in MAP1B and devoid of endogenous protein kinases was obtained from 5 day old rat brains as described by Diaz-Nido et al. [10]. MAPs were dephosphorylated with alkaline phosphatase-coated acrylic beads (Sigma, Cat. No. P0927) as previously described by Ulloa et al. [58]. Dephosphorylated MAPs were dialyzed overnight in 12.5 mM/3-glycerophosphate, 12.5 mM MOPS (pH 7.2), 0.5 mM EGTA, 7.5 mM CI2Mg, 1 mM DTT, and back-phosphorylated with p44 mpk (UBI, Cat. No. 14-102) or by casein kinase II. This kinase was purified as indicated by Diaz-Nido et al. [10].

2.4. Electrophoresis and Western blotting

Equal amounts of protein (legends to the figures), determined using the BCA method [52], from human or rat brain homogenates or SDS-treated paired helical filaments, were fractionated by gel electrophoresis and electrically transferred to nitrocellulose filters for immunostaining with anti-MAPIB 150 antibody [57].

The reaction of the blotted protein with this monoclonal antibody was detected by incubation overnight at 4°C. After incubation, the nitrocellulose was washed with PBS containing 0.05% Tween 20 and then incubated with a peroxidase-conjugated anti-mouse antibody (Dako). After a further wash, antibody binding was detected by reaction of peroxidase with Luminol as substrate, following the indication of the supplier (Amersham UK).

2.5. Immunoelectronmicroscopy

Immunoelectronmicroscopy was performed after adsorption of the samples to electron microscopy grids and incubation with 150 antibody (1/10 dilution) against MAPIB, tau or normal mouse serum for 1 h at room temperature. After extensive washing with phosphate buffered saline (PBS) the grids were incubated with goat anti-mouse antibodies conjugated with 10 nm colloidal gold particles, as previously described [36].

Fetal rat brain, adult rat brain and adult rat olfactory bulb were homogenized (1 : 1 w/v) using a teflon-glass homogenizer, in a buffer containing 20 mM Tris-HCI pH 8.0, 5 mM EGTA, 10 mM DTT, 10 mM 2-mercaptoethanol, 30 mM NaF, 1 mM sodium orthovanadate and 1 mM phenylmethylsulfonyl fluoride (PMSF). Homogenates were centrifuged 100,000×g for 1 h at 2°C, and the supernatants were collected. Brain samples of parietal and temporal cortices from AD patients and non-demented aged individuals were homogenized in the previous buffer, centrifuged at 100,000 x g for 2 min and the supernatants collected. Paired helical filaments were obtained from the pellet of 100,000 x g of Alzheimer's brain homogenate following the method of Wischik et al. [63].

Isolation of nuclear and cytoplasmic protein fractions was performed by gentle homogenization in 10 mM sodium phosphate buffer pH 7.4, 4 mM MgCl2, 2 mM EGTA, 50 mM NaF, 0.5 mM DTT, 1 mM PMSF, 10/~g/ml pepstatin, 10 ~ g / m l leupeptin and 10 mg/ml aprotinin. After centrifugation for 20 min at 1000× g, the pellet was resuspended in this buffer and centrifuged at 20,000 x g for 1 h at 2°C on a 1.5 M sucrose cushion (prepared in the same phosphate buffer). The previous supernatants (cytoplasmic fractions) were collected and the resulting pellet containing purified nuclei was resuspended in 50 mM Tris-HC1, pH 7.0, 2 mM EGTA, 50 mM NaF, 20 mM DTT and 2.5% sodium dodecyl sulfate (SDS) and boiled for 5 min. Lysed nuclei were centrifuged at 100,000x g to remove nucleic acid and the supernatant was referred to as nuclear protein fraction.

3. Results

3.1. Characterization o f M A P 1 B phosphorylation recognized by 150 antibody

A n t i b o d y 150 a g a i n s t M A P I B h a s b e e n s h o w n to

r e c o g n i z e a p h o s p h o r y l a t e d e p i t o p e [57,58] a n d t h e

m o d i f i c a t i o n o f t h a t e p i t o p e h a s b e e n p r o p o s e d to t a k e

p l a c e t h r o u g h o u t a p r o l i n e - d i r e c t e d p r o t e i n k i n a s e

( P D P K ) [57]. Fig. 1 s h o w s t h a t t h e a n t i b o d y r e a c t s w i t h

r a t m i c r o t u b u l e M A P I B p r o t e i n , d o e s n o t b i n d to

p h o s p h a t a s e - t r e a t e d M A P I B , b u t r e c o v e r s r e a c t i v i t y

w h e n t h e d e p h o s p h o r y l a t e d M A P I B is b a c k - p h o s -

p h o r y l a t e d in v i t r o w i t h a p r o l i n e - d e p e n d e n t p r o t e i n

k i n a s e s u c h as M A P k i n a s e b u t n o t w h e n it w a s m o d i -

f i ed w i t h c a s e i n k i n a s e II . A l s o , t h e r e a c t i o n w i t h 150

a n t i b o d y w a s r e c o v e r e d u p o n in v i t r o p h o s p h o r y l a t i o n

w i t h c d c 2 ( c d k l ) p r o t e i n k i n a s e ( n o t s h o w n ) .

Fig. 2 s h o w s t h a t t h e p h o s p h o M A P I B i s o f o r m s rec -

o g n i z e d b y 150 a n t i b o d y a r e m a i n l y p r e s e n t in t h e

I 1 ~ L, bTloa et al. / Molecular Brain Research 26 (1994) 113-122

AP + AP+ C AP PDPK CK2

a

L

Fig. 1. Characterization of 150 antibody raised against MAP1B. Fifty p.g of microtubule protein MAP1B from fetal rat brain untreated (C), treated with alkaline phosphatase (AP) or first treated with alkaline phosphatase, followed by back-phosphorylation with a PDPK (AP+PDPK) or CK 1I (AP+CK II), was subjected to gel electrophoresis, electroblotted onto nitrocellulose paper and probed with 150 antibody against MAPIB. The electrophoretic mobility of MW markers is indicated.

F A 0

d . . . . . . . . .

Fig. 2. Interaction of 150 antibody with MAP1B from different sources. Fifty /xg of protein from fetal (F) or adult (A) rat brain microtubules or rat olfactory bulb cell extract (O) was fractionated and processed as described in Fig. 1. The reaction of the 150 antibody with the fractionated proteins is indicated.

M A P I B -,.. P 2 2 i i ....

I 2 3 4 C A P

MAP I B . - . . . . .

P 2 2 8 - ~

Fig. 3. Presence of aberrant phosphorylated MAP1B forms in AD. The reaction of 150 antibody is shown with the cytoplasmic fraction from newborn rat brain (50 p,g) (1), the nuclear fraction from newborn rat brain (50 tzg) (2), the cytoplasmic (50 ~zg) fraction from adult rat brain (3) and the nuclear fraction (50 ~g) from adult rat brain (4). In addition, the reaction of 150 antibody is indicated with the proteins (100 ~g) of a brain cell extract from a non-demented human control (C), that of an AD patient (A) and with aggregates of paired helical filaments from AD brain (P).

Fig. 4. Presence of phosphoMAPIB in aberrant brain structures. Panel 1: nuclear staining (arrows) by 150 antibody in the insular cortex of a non-demented aged brain. No thionin counterstaining. Panel 2: insular cortex of an Alzheimer brain immunoreacted with 150 antibody in parallel with the control brain shown in panel 1. In addition to the very light nuclear staining (arrowheads), two neurofibrillary tangles (large arrows) and abundant neuropil threads (small arrows) are observed. No thionin counterstaining. Panel 3: nuclear staining (large arrows) by 150 antibody in CA 1 hippocampal cortex of a non-demented aged brain. Cytoplasmic labeling (small arrows) is due to thionin counterstaining. Panel 4: CA 1 hippocampal cortex of an Alzheimer's disease brain immunoreacted with 150 antibody in parallel to the control brain shown in panel 3, and counterstained with thionin. In neurofibrillary tangles, two types of labeling are observed, a strong granular cytoplasmic staining (open arrows) and an intense labeling of neurofibrillary degeneration (large arrows). Neuropil threads (small arrows) showed only the latter type of labeling with this antibody. Nuclear staining can be seen in neurons, bearing neurofibrillary tangles and showing granular cytoplasmic staining, (blackstars). Panel 5: neurofibrillary tangles (large arrows) and a few neuropii threads (small arrow) positive for 423 antibody are observed in an Alzheimer's disease brain. Thionin counterstaining. Panel 6: as compared with panel 5, a much more abundant number of neuropil threads (arrows) are positive for 150 antibody in an Alzheimer brain. Thionin counterstaining. Bar in panel 5 = 5 mm for the whole figure.

L. Ulloa et a l . / Molecular Brain Research 26 (1994) 113-122 117

protein from fetal rat brain microtubules, but not in adult rat brain microtubules. In adult rat brain, a positive reaction was only found in olfactory bulb, a region where neuronal regeneration continues even in the adult stage.

3.2. Presence of phosphorylated MAP1B isoforms in the brains of AD patients

In previous reports, there is some controversy about the differences in the amount of MAP1B in AD brains

O ~ m

" 6

a

B

0 4 ~,

0

~m

m )iiii

m a

I

e A

kC... . 8

!

P

)

~0 ir

3~ r 4 " ~

/

B

1 w

, - - ' 5 ! ~'" ~%

%

t ~

w

o

%

I I

2

118 L. Ulloa et aL / Molecular Brain Research 26 (1994) 113-122

compared to that of controls. Two reports indicate an increased amount of MAPIB in AD [19,53], but a third describes a slightly decreased amount of MAP1B in AD [41]. In all cases, the measurement of MAP1B was based on the use of specific, but different, antibodies to MAP1B. Thus, a possible explanation of these differences may be the measurement of different MAPIB isoforms, caused by the use of different antibodies in each case. Based in their mode of phosphorylation, two different classes of MAP1B isoforms have been described; mode I-phosphorylated MAP1B isoforms can be identified by their specific reaction with 150 antibody, whereas mode II phosphoisoforms are recognized by antibody 125 or by that used in the previous report [41]. To test whether the reported increase in MAP1B is due mainly to an increase in mode I phos- phoMAP1B isoforms, 150 antibody was used in West- ern blot as shown in Fig. 3.

Fig. 3 shows that 150 antibody reacts with a protein with the electrophoretic mobility of rat brain microtubule MAP1B, present in AD brain extracts but not in non-demented aged brain extracts. In the latter extracts, the antibody reacts with a faster-migrating protein with a MW of 220 kDa also present in AD brain fractions. The 220 kDa protein appears to be a nuclear protein, related to MAPIB, as also indicated in Fig. 3, which shows that, in rat brain, it appears specifically in the nuclear, but not in the cytoplasmic fractions. On the other hand, Fig. 3, shows no reaction, at the conditions used, with proteins moving with the electrophoretic mobility of tau.

When antibody 125 [58] was used no increase in the reaction of that antibody with the protein isolated from AD respect to that from non-demented aged brain extracts was observed (data not shown).

For further localization of MAPIB in AD brain, bundles of paired helical filaments were isolated as described by Wischik et al. [63] and the presence of MAP1B, in that fraction tested. Fig. 3 shows that MAPIB but not the 220 kDa protein, is enriched in the fraction containing aggregates of paired helical filaments.

3.3. Distribution of phosphorylated MAPIB isoforms identified by 150 antibody in AD brain

All types of structures with neurofibrillary degeneration (NFT, NT a n d / o r plaque-associated neurites) were stained by the anti-tau 423 antibody in AD brains, confirming previous observations [42]. An example of NFT and NT stained by 423 antibody is shown in Fig. 4, panel 5.

150 antibody also labeled all the structures undergo- ing neurofibriUary degeneration. In every case, 150 antibody stained these structures in a higher proportion than the anti-PHF tau antibody used. This was

especially evident for NT, which number observed with the 150 antibody was more than ten-fold that shown with the 423 antibody (Fig. 4, panels 5 and 6). Addi-

Fig. 5. Association of phospho MAPIB to PHF aggregates. PHF were purified as described by Wischik et al. (1986) and the immunoelectron microscopy analyses using 150 antibody (A), the polyclonal tau antibody (B) and the normal mouse serum (C) are indicated.

L. Ulloa et a l . / Molecular Brain Research 26 (1994) 113-122 119

tionally, a strong granular cytoplasmic labeling was observed with the 150 antibody, (Fig. 4, panel 4).

In addition to strongly-stained neurofibrillary degeneration, 150 antibody labeled abundant nuclei throughout the cortex in non-demented aged (see panels 1 and 3 of Fig. 4) and, more lightly, in AD brains (Fig. 4, panels 2 and 4). Sections counterstained with thionin allowed the identification of most of these labeled nuclei as belonging to pyramidal neurons (Fig. 4, panels 3 and 4). These results are in agreement with recent publications indicating that 150 antibody stains brain cells [32,45,46,47].

Pretreatment with alkaline phosphatase resulted in a dramatic decrease in the number of NT stained by the 150 antibody, whereas the nuclear staining showed a moderate lowering. However, alkaline phosphatase only slightly decreased the number of neurons with NFT stained with 150 antibody, without modifying the granular cytoplasmic labeling (data not shown).

Antibody 125 (against mode II phosphoMAP1B) stained only a very low proportion, if any, of structures with neurofibrillary degeneration (data not shown).

3.4. Presence of phosphoMAP1B in neurofibrillary tangles

As described above, phosphoMAPIB isoforms which can be recognized by 150 antibody are associated to neurofibrillary tangles. Since tangles are composed of PHF bundles, these structures have been purified to test whether or not MAP1B is a major component of these filaments. Fig. 5 shows, in immunoelectron microscopy analysis, a slight reaction of 150 antibody with isolated PHF and a stronger reaction with PHF aggregates (NFT). A positive reaction for PHF was found when an anti-tau antibody was used (positive control), but normal mouse serum (negative control) did not react with PHF, suggesting that phosphoMAP1B is not a major component of PHF and that it probably associ- ates with them when they form aggregates or NFT.

4. Discussion

Microtubule-associated proteins MAP1B and tau appear to be present in two pathological structures, NFT and dystrophic neurites (DN), either isolated in the neuropil as NT or associated to senile plaques. Some studies support a positive correlation between severity of dementia and accumulation of NFT in different brain areas [1,9,30,31,44,62]. Less is known about DN, although an even more positive correlation has been demonstrated between these pathological structures and severity of dementia [30,31,34]. In fact, it has been suggested that DN precede NFT formation

[3,29,30,31] being their presence an early indication of AD [30,31].

In a manner similar to that indicated for tau, which has been found to be hyperphosphorylated when present in aberrant structures such as NFT [2,17,18], MAP1B is also aberrantly phosphorylated when it is present in neurofibrillary tangles (in which it appears to be only an associated protein) and in DN [19,53]. Additionally, a phosphoMAP1B-related protein, with a MW (220 kDa) lower than that of MAP1B, was found to be present in non-demented aged and in AD brains. This protein appears to be a nuclear protein as determined by subcellular fractionation and immunocytochemical analyses [58].

The fact that pretreatment with alkaline phosphatase dramatically decreased the staining for 150 antibody in NT agrees well with the already demonstrated specificity of this antibody for growth cones [32], thus supporting the possibility that an important number of NT may be related with sprouting events [19].

Two modes of phosphorylation have been described for MAP1B, one (mode I) in which a proline-directed protein kinase (PDPK) is involved and the other (mode II) in which casein kinase II (CKII) modifies the protein [57,58]. Mode I occurs only during early developmental stages and has been correlated with neurite outgrowth [32,58] and developing axonal processes. This feature is compatible with a previous hypothesis [19] which suggests that the modification of MAPIB associated with dystrophic neurites and neurofibrillary tangles in AD may be correlated with the neurite outgrowth that occurs in patients with this disorder. Here it is shown that the modification appears to be specific for a PDPK phosphorylation. An aberrant PDPK phosphorylation has also been suggested for other proteins such as tau, in which phosphorylated fetal forms have been found in AD [7,13]. Also, in AD aberrantly phosphorylated MAP1B isoforrns are those which are nor- mally expressed only in early developmental stages, as indicated in Fig. 2. Thus, aberrant phosphorylation, similar to that which takes place at fetal stages, by a PDPK, appears to be a common feature for the modification of tau, the major component of PHF, and for MAPIB, a component of dystrophic neurites, and also an NFT-associated protein. A simple model of this aberrant phosphorylation of microtubule-associated proteins can be proposed. It implies that both tau and MAPIB could be aberrantly phosphorylated by PDPK, and that those aberrant phosphorylated proteins are components of the neurofibrillary degeneration in AD.

It thus appears very important to identify the PDPK involved in this aberrant phosphorylation. It may corre- spond to a MAP kinase since both tau [11] and MAPIB may be modified in vitro, by these kinases. However, MAP kinases are expressed throughout different devel-

12[) L. Ulloa et al . /Molecular Brain Research 26 (1994) 113-122

opmental stages [5], whereas cdc-2-1ike kinases are expressed mainly at fetal stages [20]. It should there- fore be pointed out that the amount of a brain cdc-2- like kinase, mainly expressed at fetal stages, is increased in cell homogenates of AD brain [28,66]. It should also be indicated that PDPK phosphorylation in tau [11] and MAP1B could be reversed by phosphatase PP2A [14,59] a phosphatase whose activity is lower in AD than in control brains [15].

Acknowledgments

The authors want to thank Drs. C. Wischik and J. Xuereb (Cambridge Brain Bank) for providing brain samples, Dr. Kosik, Dr. Amalia S~inchez and Dr. J. Diaz-Nido for helpful suggestions, and M. Vicenta G6mez and Milagros Guerra for technical assistance. We appreciate the collaboration of Dr. Carmen Navarro (Meixoeiro Hospital, Vigo, Spain), Dr. Ana Cabello and Dr. Santiago Madero (12 de Octubre Hospital, Madrid, Spain) and Dr. Elliot Mufson (Rush-Presbyterian-St. Luke's Medical Center, Chicago IL, U.S.A.), for providing some of the human tissue used in the study. This work was supported by the Fondo de Investigaciones Sanitarias de la Seguridad Social 93/0198 (Spain) and by a grant from CICYT and the Direcci6n General Investigaciones Cientlficas y T6cnica (Spain). The institutional support of the Fundaci6n Ram6n Areces to C.B.M. is also acknowl- edged.

Abbreviations

AD CKII DN MAP NFT PHF PDPK

Alzheimer's disease casein kinase II dystrophic neurites microtubule associated protein neurofibrillary tangles paired helical filaments proline directed protein kinase

References

[1] Arriagada, P.V., Growdon, J.H., Hedley-Whyte, T. and Hyman, B.J., Neurofibrillary tangles but not senile plaques parallel dura- tion and severity of Alzheimer's disease, Neurology, 42 (1992) 631-639.

[2] Bancher, C., Brunner, C., Lassmann, H., Budka, H., Jellinger, K., Wiche, G., Seitelberger, F., 17,18, I., Iqbal, K. and Wis- niewski, H.M., Accumulation of abnormally phosphorylated tau precedes the formation of neurofibrillary tangles in Alzheimer's disease, Brain Res., 477 (1989) 90-99.

[3] Benzing, W.C., Ikonomovic, M.C., Brady, D.R., Mufson, E.J. and Armstrong, D.M., Evidence that transmitter containing

dystrophic neurites precede paired helical filament and Alz50 formation within senile plaques in the amygdala of non-demented elderly and patients with Alzheimer's disease, J. Comp. Neurol., 334 (1993) 176-191.

[4] Bloom, G.S., Luea, F.C. and Vallee, R.B. Microtubule-associated protein MAP1B. Identification of a major component of the neuronal cytoskeleton, Proc. Natl. Acad. Sci. USA, 82 (1985) 5404-5408.

[5] Boulton, T.G., Nye, S.H., Robbins, A., Ip, N.Y., Radziejewska, E., Morgendeser, S.D., de Pinho, R.A., Panayotatos, N., Cobb, M.H. and Yancopoulos, G.D., ERKS: a family of protein- serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF, Cell, 65 (1991) 663-675.

[6] Braak, H., Braak, E., Grundke-Iqbal, I. and lqbal, K. Occur- rence of neuropil threads in senile brain and in Alzheimer's disease. A third location of paired helical filaments outside of neurofibrillary tangles and neuritic plaques, Neurosci. lett., 65 (1986) 351-355.

[7] Bramblett, G.T., Goedert, M., Jakes, R., Mernick, S.E., Tro- janowski, J.Q. and Lee, V.M.Y., The abnormal phosphorylation of tau at Ser 396 in Alzheimer's disease recapitulates phosphorylation during development and contributes to reduced microtubule binding, Neuron, 10 (1993) 1089-1099.

[8] Busciglio, J., Ferreira, A., Steward, O. and Caceres, A. An immunocytochemical and biochemical study of the microtubule associated protein tau during postlesion afferent reorganization in the hippocampus of adult rat, Brain Res., 419 (1987) 244-252.

[9] Dela~re, P., Duyckaerts, C., Brion, J.P., Poulain, V. and Hauw, J.J., Tau, paired helical filaments and amyloid in the neocortex: a morphometric study of 15 cases with graded intellectual status in aging and senile dementia of Alzheimer's type, Acta Neu- ropathol., 77 (1989) 645-653.

[10] Diaz-Nido, J., Serrano, L., M~ndez, E. and Avila, J., A casein kinase II related activity is involved in phosphorylation of microtubule associated protein MAPIB during neuroblastoma cell differentiation, J. Cell Biol., 106 (1988) 2057-2065.

[11] Drewes, G., Lichtenberg-Kraag, B., Doring, F., Mandelkow, E.M., Biernat, J., Gonis, J., Doree, M. and Mandelkow, E., Mitogen activated protein (MAP) kinase transforms Tau protein into an Alzheimer-like state, EMBO J., 11 (1992) 2131-2138,

[12] Fisher, I. and Romano-Clarke, G.C., Association of microtubule associated protein MAP1B with growing axons in cultured hippocampal neurons, Mol. Cell. Neurosci., 2 (1991) 39-51.

[13] Goedert, M., Jakes, R., Crowther, R.A., Six, J. Lubke, U., Vandermeeren, M., Cras, P., Trojanowski, J.Q. and Lee, V.M.Y., The abnormal phosphorylation of tau protein at ser 202 in Alzheimer disease recapitulates phosphorylation during development, Proc. Natl. Acad. Sci. USA, 90 (1993) 5066-5070.

[14] Goedert, M., Cohen, E.S., Jakes, R. and Cohen, P., P42 MAP kinase phosphorylafion sites in microtubule associated protein tau are dephosphorylated by protein phosphatase 2A1: implica- tions for Alzheimer's disease, FEBS Lett., 312 (1992) 95-99.

[15] Gong, C.X., Singh, T., Grundke-Iqbal, I. and Iqbal, K., Phos- phoprotein phosphatase activities in Alzheimer disease brain, J. Neurochem., 61 (1993) 921-927.

[16l Gonz~ilez, P.J., Correas, I. and Avila, J., Solubilization and fraetionation of paired helical filaments, Neuroscience, 50 (1992) 491-499.

[17] Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y.C., Zaidi, M.S. and Wisniewski, H.M., Microtubule-associated protein tau: a component of Alzheimer paired helical filaments, J. Biol. Chem., 261 (1986) 6084-6089.

[18] Grundke-Iqbal, I., Iqbal, K., Tung, Y.C., Quinlan, M., Wis- niewski, H.M. and Binder, L.I., Abnormal phosphorylation of the microtubule-associate protein tau in Alzheimer cytoskeletal pathology, Proc. Natl. Acad. Sci. USA, 83 (1986) 4913-4917.

L. Ulloa et al. / Molecular Brain Research 26 (1994) 113-122 121

[19] Hasegawa, M., Arai, T. and Ihara, Y., Immunochemical evidence that fragments of phosphorylated MAP5 (MAP1B) are bound to neurofibrillary tangles in Alzheimer's disease, Neuron, 4 (1990) 909-918.

[20] Hayes, T.E., Valtz, M.L. and Mckay, R.D., Down regulation at cdc2 upon terminal differentiation of neurons, New Biol., 3 (1991) 259-269.

[21] Ihara, Y., Massive somatodendritic sprouting of cortical neurons in Alzheimer's disease, Brain Res., 459 (1988) 138-144.

[22] Ihara, Y., Nukina, N., Miura, R. and Ogawara, M., Phosphory- lated tau protein is integrated into paired helical filaments in Alzheimer's disease, J. Biochem., 99 (1986) 1807-1810.

[23] Karr, T.L., White, H.D. and Purich, D.L., Characterization of brain microtubule proteins were prepared by selective removal of mitochondrial and synaptosomal components, J. Biol. Chem., 254 (1979) 6107-6111.

[24] Khatchaturian, Z.S., Diagnosis of Alzheimer's disease, Arch. Neurol., 42 (1985) 1097-1105.

[25] Kosik, K., Alzheimer's disease a cell biological perspective, Science, 256 (1992) 780-783.

[26] Kosik, K.S., Joachim, C.L. and Selkoe, D.J., Microtubule-associated protein tau is a major antigenic component of paired helical filaments in Alzheimer's disease, Proc. Natl. Acad. Sci. USA, 83 (1986) 4044-4048.

[27] Kowall, N.W. and Kosik, K.S., Axonal disruption and aberrant localization of tau protein characterize the neuropil pathology of Alzheimer's disease, Ann. Neurol., 22 (1987) 639-643.

[28] Ledesma, M.D., Correas, I., Avila, J. and Diaz-Nido, J., Impli- cation of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease, FEBS Lett., 308 (1992) 218-224.

[29] Lenders, M.B., Peers, T.C., Tramu, G., Delacourte, A., Defos- sez, A., Petit, H. and Mazzuca, M., Dystrophic peptidergic neurites in senile plaques of Alzheimer's disease hippocampus precede formation of paired helical filaments, Brain Res., 481 (1989) 344-349.

[30] McKee, A.C., Kosik, K.S. and Kowall, N.W., Neuritic pathology and dementia in Alzheimer's disease, Ann. Neurol., 30 (1991) 156-165.

[31] Mckee, A.C., Kosik, K.S. and Kowall, N.W., Dystrophic neurites precede neurofibrillary tangle formation and mark the progres- sion of dementia in Alzheimer's Disease, J. Neuropathol. Exp. Neurol., 50 (1991) 315-325.

[32] Mansfield, S.G., Diaz-Nido, J., Gordon Weeks, P.R. and Avila J., The distribution and phosphorylation of the microtubule associated protein MAP1B in growth cones, J. Neurocytol., 21 (1992) 1007-1022.

[33] Masliah, E., Mallory, M., Hansen, L., Alford, M. Albright, T., De Teresa, R., Terry, R., Baudier, J. and Saitoh, T., Patterns of aberrant sprouting in Alzheimer's disease, Neuron, 6 (1991) 729-739.

[34] Masliah, E., Ellisman, E., Carragher, B., Mallory, M., Young, S., Hansen, L., De Teresa, R. and Terry, R.D., Analysis of the relationship between synaptic pathology and neuropil threads in Alzheimer's disease, J. Neuropathol. Exp. Neurol., 51 (1992) 404-414.

[35] Mena, R., Wischik, C.M., Novak, J., Milstein, C. and Coello, C.A., Progressive deposition of paired helical filaments (PHF) in the brain characterized the evolution of dementia in Alzheimer's disease. An immunocytochemical study with monoclonal antibodies against the PHF case, J. Neuropathol. Exp. Neurol. 50 (1991) 474-490.

[36] Montejo de Garcini, E., Carrascosa, J.L., Nieto, A. and Avila, J., Collagenous structures present in brain contains epitopes shared by collagen and microtubule associated protein tau, J. Struct. Biol., 103 (1990) 34-39.

[37] Montejo de Garcini, E., Diez J.C. and Avila, J. Quantitation and characterization of tau factor porcine tissues, Biochim. Biophys. Acta, 881 (1986) 456-461.

[38] Mor:in, M.A. and G6mez-Ramos, P., Cholinesterase histochem- istry in the human brain: effect of various fixation and storage conditions, J. Neurosci. Methods, 43 (1992) 49-54.

[39] Mori, M., Kondo, J., Kondo, J. and Ihara, Y., Ubiquitin is a component of paired helical filaments in Alzheimer's disease, Science, 235 (1987) 1641-1644.

[40] Morishima-Kawashima, M., Arai, T., Ogawara, M., Takio, K., Titani, K., Saitoh, T., Kosik, K.S. and Ihara, Y., A possible fetal antigen of Mr. 70000 in neurofibrillary tangles, Brain Res., 554 (1991) 316-320.

[41] Nieto, A., Montejo de Garcini, E. and Avila, J., Altered levels of microtubule proteins in brains of Alzheimer's disease patients, Acta Neuropathol., 78 (1989) 47-51.

[42] Novak, J., Jakes, R., Edwards, P.C., Milstein C. and Wischik, C.M., Differences between tau protein of Alzheimer's paired helical filaments case and normal tau revealed by epitope analyses of monoclonal antibodies 423 and 7.51, Proc. Natl. Acad. Sci. USA, 88 (1991) 5837-5841.

[43] Peterson, C., Kress, Y., Vallee, R.B. and Goldman, J.E., High molecular weight microtubule associated proteins bind to actin lattices (Hirano bodies), Acta NeuropathoL, 77 (1988) 168-174.

[44] Price, J.C., Dawis, P.B., Morris, J.C. and White, D.L., The distribution of tangles, plaques and related immunohistochemi- cal marks, in healthy aging and Alzheimer's disease, Neurobiol. Aging, 12 (1991) 295-312.

[45] Riederer, B., Cohen, R. and Matus, A., MAP5 a novel brain microtubule associated protein under strong developmental regulation, J. Neurocytol., 15 (1985) 763-775.

[46] Riederer, B.M., Moya, F. and Calvert, R., Phosphorylated MAP1B, also MAP5 and MAP1X, is involved in axonal growth and neuronal mitosis, NeuroReport, 4 (1993) 771-774.

[47] Riederer, B.M., Guadafio-Ferraz, A. and Innocenti, G.M., Dif- ference in distribution of microtubule associated proteins 5a and 5b during the development of cerebral cortex and corpus callosum cats: dependence on phosphorylation, Dec. Brain Res., 56 (1990) 235-243.

[48] Sato-Yoshitake, R., Shiomma, Y., Miyasaka, H. and Hirokawa, N., Microtubule associated protein MAP1B molecular structure, localization and phosphorylation dependent expression in developing neurons, Neuron, 3 (1989) 229-238.

[49] Schoenfeld, T.A., Mckenacher, L., Obar, R. and Vallee, R.B., MAP1A and MAP1B are structurally related microtubule associated proteins with distinct developmental patterns in the CNS, J. Neurosci., 9 (1989) 1712-1730.

[50] Shelanski, M., Gaskin, F. and Cantor C.R., Microtubule assem- bly in the absence of added nucleotides, Proc. Natl. Acad. Sci. USA, 70 (1973) 765-768.

[51] Selkoe, D.J., The molecular pathology of Alzheimer's disease, Neuron, 6 (1991) 487-498.

[52] Smith, P.K., Krohn, R.I., Hermanson, E.T., Mallia, A.K., Gart- ner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150, (1985) 76-85.

[53] Takahashi, H., Hirokawa, K., Ando, S. and Obata, K., Immuno- histological study on brains of Alzheimer's disease using antibodies to fetal antigens, C-series gangliosides and microtubule associated protein 5, Acta Neuropathol., 81 (1991) 623-631.

[54] Terry, R.D., Peck, A., De Teresa, R., Schechter, R. and Honou- pian, D.S., Some morphometric aspects of the brain in senile dementia of the Alzheimer type, Ann. Neurol., 10 (1981) 184- 192.

[55] Tomlinson, B.E., Blessed, G. and Roth, M., Observation of the brain of demented old people, J. Neurol. Sci., 11 (1970) 205-242.

122 L. Ulloa et al. / Molecular Brain Research 26 (1994) 113-122

[56] Tucker, R.P., Garner C.C. and Matus A., In situ localization of microtubule associated protein in RNA in the developing and adult rat brain, Neuron, 2 (1989) 1245-1246.

[57] Ulloa, L., Diaz-Nido, J. and Avila, J., Depletion of casein kinase II by antisense oligonucleotide prevents neuritogenesis in neuroblastoma cells, EMBOJ., 12 (1993)1633-1640.

[58] Ulloa, L., Avila, J. and Diaz-Nido, J., Heterogeneity in the phosphorylation of microtubule associated protein MAP1B during rat brain development, J. Neurochem., 61 (1993) 961-972.

[59] Ulloa, L., Dombradi, V., Diaz-Nido, J., Sziias, K., Gergely, P., Friedrich, P. and Avila, J., Dephosphorylation of distinct sites on microtubule associated protein MAP1B by protein phos- phatases 1, 2A and 2B, FEBS Lett., 330 (1993) 85-89.

[60] Viereck, C., Tucker, R.P. and Matus, A., The adult rat olfactory system expresses microtubule associated proteins found in the developing brain, J. Neurosci., 9 (1989) 3547-3557.

[61] Watson, Jr. R.E., Wiegand, S.J., Clough, R.W. and Hoffman, G.E., Use of cryoprotectant to maintain long-term peptide im- munoreactivity and tissue morphology, Peptides, 7 (1986) 155- 159.

[62] Wilcock, G.K. and Esiri, M.M., Plaques, tangles and dementia: a quantitative study, J. Neurol Sci., 56 (1982) 343-356.

[63] Wischik, C.M., Crowther, R.A., Stewart, M. and Roth, M., Subunit structure of paired helical filaments in Alzheimer's disease, J. Cell Biol., 100 (1985) 1905-1912.

[64] Wischik, C.M., Novak, M., Thorgersen, H.C., Edwards, P.C., Runswik, M.J., Jakes, R., Walker, J.E., Milstein, C., Roth, M. and Klug, A., Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease, Proc. Natl. Acad. Sci. USA, 85 (1988) 4506-4510.

[65] Wood, J.G., Mirra, S.S., Pollock, N.J. and Binder, J.I., Neurofib- rillary tangles of Alzheimer's disease share antigenic determi- nants with the axonal microtubule-associated protein tau, Proc. NatL Acad. Sci. USA, 83 (1986) 4040-4043.

[66] Wood, J.G., Lu, Q., Reich, C. and Zinsmeister, P., Proline-directed kinase systems in Alzheimer's disease pathology, Neu- rosci. Lett., 156 (1993) 83-86.

microtubule-associated protein map1b showing a fetal phosphorylation pattern is present in sites of...

Documents