Distribution of phosphate-independent MAP2 epitopes revealed with monoclonal antibodies in microwave-denatured human nervous system tissues

Journal of Neuroscience Methods, 29 (1989) 171-180 171 Elsevier

NSM 00976

Distribution of phosphate-independent MAP 2 epitopes revealed with monoclonal antibodies

in microwave-denatured human nervous system tissues

John Q. Trojanowski, Theresa Schuck, M. Luise Schmidt and Virginia M.-Y. Lee Department of Pathology and Laboratory Medicine (Neuropathology), University of Pennsylvania School of Medicine,

Philadelphia, PA 19104-6079 (U.S.A.)

(Received 17 October 1988) (Revised 3 March 1989)

(Accepted 6 March 1989)

Key words: M i c r o t u b u l e a s soc i a t ed p r o t e i n - 2 ; N e u r o f i l a m e n t ; G l i a l f i l a m e n t ; M i c r o w a v e d e n a t u r a t i o n

In contrast with results obtained in experimental animals, antibodies to microtubule associated protein-2 (MAP2) preferentially label abnormal structures in human nervous system tissue samples, but the normal sites at which MAP 2 is expressed are not well-defined. To determine the distribution of MAP 2 in the human central (CNS) and peripheral (PNS) nervous systems, we prepared monoclonal antibodies (MAbs) specific to MAP 2, and compared the localization of this MAP in postmortem bovine and human tissues as well as in several human neural cell lines that express either neurofilament (NF) or glial filament (GF) proteins. Eight MAbs specific for phosphate-independent epitopes in bovine and human MAP 2 were obtained, and those that performed well in tissues produced immunoreactivity confined to the somatodendritic domain of neurons in bovine and human CNS and PNS tissues. Other neural cells (e.g. astrocytes) did not express MAP 2 immunoreactivity using these MAbs. Postmortem delays of < 24 h prior to tissue denaturation did not affect the distribution of MAP 2 immunoreactivity. However, microwave denaturation of these tissues preserved MAP 2 immunoreactivity better than fixation with Bouin's solution or formalin. Microwave treatment also improved the immunoreactivity of several MAbs for NF and GF proteins. Finally, MAP 2 was not detected in human neural cell lines that express NF (2) or GF (1) proteins. We conclude that microwave denaturation provides an effective means to preserve the immunoreactivity of normal human neuronal cytoskeletal proteins, and that this method of tissue denaturation allows the normal distribution of MAP 2 to be defined in postmortem samples of human CNS and PNS tissues.

Introduction

T h e res t r i c t ed i m m u n o c y t o c h e m i c a l loca l i za -

t i on o f cy toske l e t a l p ro t e in s in n e u r o n s o f exper i -

m e n t a l a n i m a l s has b e e n sugges t ed to p r o v i d e a

m o l e c u l a r bas is fo r the e s t a b l i s h m e n t o f n e u r o n

a s y m m e t r y a n d fo r the l i f e - long s tab i l i ty o f h i g h l y

e l o n g a t e d a n d ex t ens ive ly b r a n c h e d d e n d r i t e s a n d

Correspondence: J.Q. Trojanowski, Division of Neuropa- thology, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6079, U.S.A.

axons which often comprise the overwhelming mass of a neuron (see reviews by Olmstead, 1986; Schlaepfer, 1987; Matus, 1988). For example, the somatodendritic domain of a neuron is the region in which the microtubule associated protein-2 ( M A P ) , M A P 2 (see M a t u s , 1988 a n d re fe rences

there in) , a n d p o o r l y p h o s p h o r y l a t e d i s o f o r m s of

t he h igh a n d m i d d l e m o l e c u l a r w e i g h t ( M r) n e u r o -

f i l a m e n t ( N F ) s u b u n i t s ( N F - H a n d N F - M , respec-

t ively) ( S t e r n b e r g e r a n d S t e rnbe rge r , 1983; L e e e t

al., 1987) a r e p r e f e r e n t i a l l y expressed . N e v e r t h e -

less, s o m e M A P 2 i s o f o r m s m a y be e x p r e s s e d in

gl ia ( P a p a s o z o m e n o s a n d Binder , 1986). C u r -

ren t ly , l i t t le is k n o w n a b o u t the n o r m a l d i s t r ibu -

0165-0270/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

172

tion of MAP 2 in human brain. Indeed, anti-MAP 2 antibodies primarily label abnormal structures in postmortem tissues (Kosik et al., 1984; Yen et al., 1987).

To gain insights into the normal biology of human MAP2, we prepared monoclonal antibodies (MAbs) using bovine MAP 2 immunogens, demonstrated their specificity for human and bovine MAP2, and used them to probe the distribution and immunological stability of this MAP in human tissues denatured by a variety of methods. These data were compared with data obtained from studies of bovine tissue samples which were processed and probed in the same manner. We also assessed whether or not MAP2 was detec- table in several putative neuronal and glial human cell lines that express NF or glial filament (GF) proteins, respectively.

Materials aml Methods

Human and bovine tissue preparation Bovine brains were obtained immediately after

sacrifice in a uniform manner (bolt-gun) from a slaughterhouse, and transported to the laboratory on ice or in fixative. Postmortem intervals (PMIs) of 30 rain and 1-4 , 6 -8 , 11, 12, 20, 24 and 30 h were achieved by keeping the tissue at 4°C in a humidified vessel. Samples of postmortem human CNS and PNS tissues were obtained from over 40 subjects with and without evidence of neurological disease. The criteria for diagnosis, and the sam- pling procedures have been described (Schmidt et al., 1987, 1988a,b). Five subjects ranged in age from 2 days to 30 years old, and the remainder were 50-94 years old. An equal mix of genders was studied, The PMI ranged from 3 to 24 h.

Tissues were either chemically fixed or denatured by microwave treatment. Chemically fixed tissues were immersed in fixative, and trimmed to 3-4 mm thick slabs at 30-60 min intervals to facilitate penetration by 10% neutral buffered formalin or Bouin's solution. Bouin's solution had a final pH of 3.0 and consisted of: 71% (v/v) saturated picric acid (Baker); 24~ (v/v) stock (37%) formaldehyde (Baker); 5% (v/v)glacia l acetic acid (Baker); and 0.7% (w/v) NaC1. Irnmer-

sion in fixative was terminated after it penetrated the tissue blocks (4-18 h) by washing the tissue in several changes of Tris-salme buffer (TBS), pH 7.6.

Chemically fixed tissues were embedded in paraffin and 6 ~tm-thick sections were cut on a rotary microtome. Some samples of Bouin's fixed bovine tissues were cut at 30 #m as unembedded blocks with a vibrating microtome (Dosaka DTK- 3000, Japan). Paraffin embedding was as follows: 70% alcohol (3 h), 80% alcohol (1 h), 2 changes of 95% alcohol (1 and 2 h each), 3 changes of 100% alcohol (2, 1.5 and 1.5 h each), 3 changes of xylene (1 h each) and 2 changes of paraffin (1.5 h each). The solvents were at room temperature, and the paraffin at 60 o C.

Other blocks of bovine hippocampus and human CNS and PNS sites were immersed in TBS and exposed to microwave irradiation. Briefly, 5-10 ram-thick blocks of tissue were placed in 120 ml plastic beakers, brought to a volume of 60 ml with TBS at 22°C, and 14 beakers were arranged at the periphery of a rotating carousel in a microwave oven (Riccar-RM-1280) and microwaved for 7 min (power level 8) which raised the TBS to 68°C + 3°C. These blocks were transferred to 70% alcohol for paraffin embedding and section- hag as described above.

Human cell lines Three cell lines derived from 3 different human

medulloblastomas and 1 cell line derived from a human glioma also were examined. Two of the medulloblastoma cell lines expressed NF triplet proteins (D283 MED, D341 MED), the other one (DAOY) did not, and none of these cell lines expressed GF protein (see He et al., 1989 and references therein). The human ghoma cell line (U251 MG) expressed GF, but not NF triplet proteins (Bigbee et al., 1983; Trojanowski et al., unpublished). Pelleted cells (5-10× 106 ) from each line were prepared and probed with MAbs as described elsewhere (He et al., 1989).

Imrnunoperoxidase methods Sections affixed to poly-L-lysine (Sigma) coated

slides (5 #1 of a 1% solution/slide) were air-dried or incubated in an oven at 45°C for 12-16 h,

p r o b e d wi th M A b s using the a v i d i n - b i o t i n m e t h o d as prev ious ly descr ibed (Ca rden et al., 1987; Lee et al., 1987; Schmid t et al., 1987, 1988), and coun te r s t a ined br ie f ly wi th hematoxy l in . A d - di t ionaUy, we examined the in situ p h o s p h o r y l a - t ion s tate of M A P 2 ant igens recognized b y our M A b s using Escherichia coli alkaline p h o s p h a t a s e (Type I I I -N , Sigma) t r ea tmen t of the sect ions p r io r to the app l i ca t ion of the M A b s as desc r ibed ear l ier (Lee et al., 1987; Schmid t et al., 1987).

Production and immunochemical characterization of anti-MAP 2 MAbs

M A P 2 pro te ins used for i m m u n i z a t i o n and screening of M A b s were i so la ted b y p rocedures

M- ! -

i i

A B C D Fig. 1. Immunoblots of enriched bovine (A,B) and human (C,D) MAP 2 (M) and tau (T) proteins. The anti-MAP 2 MAb (M20) was applied to the nitrocellulose strips in A and B before (A) and after (B) Enzymatic dephosphorylation with E. coli alkaline phosphatase, and the MAP 2 bands axe relatively unaffected by this treatment. The MAb in C (T46), which is not listed in Table I, cross-reacts with both human MAP 2 and tan, while the MAb in D (M12) binds only to human MAP 2. When these and the other anti-MAP 2 MAbs were screened against bovine and human spinal cord fractions enriched in NF and GF proteins as described elsewhere (Lee et al., 1988a,b), no cross-reactions with proteins in these fractions were noted.

173

TABLE I

PROPERTIES OF MONOCLONAL ANTIBODIES

The properties of the MAbs used in this study axe listed here according to their specificities for bovine (upper portion of table) or human (lower portion of table) MAP 2. The code for each MAb is shown in the monoclonal antibodies column. The subsequent columns provide information on the subunit specificity, and the phosphorylation state of the epitope recognized by the MAbs. A phosphate independent, P(ind), epitope refers to one that does not depend on the presence or absence of phosphates for antibody recognition as described earlier (Lee et al., 1987; 1988a,b; Schmidt et al., 1987).

Monoclonal antibodies Speci- Phosphoryl- ficity ation state

Bovine M8, M20 MAP 2 P(ind)

Human M4, M5, M8, M12, M20, M20, T31, T37, T47 MAP 2 P(ind)

s imi lar to those desc r ibed b y Binder et al. (1985). The p rocedure s for h y b r i d o m a p roduc t ion , and the E L I S A and i m m u n o b l o t me thods used to charac te r ize the M A b s have been pub l i shed (Lee et al., 1986, 1987, 1988a,b; C a r d e n et al., 1987; Schmid t et al., 1987, 1988). I m m u n o b l o t s demon- s t ra t ing the specif ic i ty of represen ta t ive M A b s for bov ine and h u m a n M A P 2 are shown in Fig. 1. Al l of the a n t i - M A P 2 M A b s used here are p h o s p h a t e i n d e p e n d e n t or P( ind) s ince they b i n d s t rongly to M A P 2 be fore a n d af te r enzymat i c d e p h o s p h o r y l a - t ion unde r cond i t ions tha t d e p h o s p h o r y l a t e N F p ro te ins and ano the r M A P , i.e. tan p ro te ins (Lee et al., 1986, 1987, 1988a,b; Schmid t et al., 1987; K o s i k et al., !988; T ro j anowsk i et al., 1989). Ad- d i t iona l a n t i - N F and a n t i - G F p ro t e in M A b s (Lee et al., 1984, 1986, 1987, 1988a,b) also were tested for their ab i l i ty to de tec t N F and G F pro te ins in mic rowave d e n a t u r e d versus Bouin ' s f ixed tissues. The a n t i - M A P 2 M A b s p r o d u c e d here and their bov ine and h u m a n specif ici t ies are shown in Tab le

I.

Results

General observations C o m p a r e d to several o ther f ixatives, Bouin ' s

so lu t ion was shown to p rov ide op t ima l preserva-

174

i i!i! ~ . :?!~

Fig. 2. Adjacent paraffin sections of subiculum-CA1 (A), CA2-CA3 (B) and dentate gyrus (C) from bovine hippocampus (PMI = 12 h) denatured by microwave irradiation and probed with an anti-MAP 2 (M20) MAb. M20 stains the soma and dendrites of neurons and other fine processes. Axons in white matter (not shown), and those traversing CA2-CA3 (arrowheads in B) are negative. All of

the photomicrographs are at the same magnification, and the scale bar in A = 100 #m.

tion of a large number of NF epitopes (Hickey et al., 1983; Trojanowski et al., 1986; Schmidt et al., 1987). We found here that MAP 2 immunoreactivity was nearly completely extinguished by formalin, while MAP 2 immtmoreactivity was preserved well in both Bouin's fixed and microwave- denatured tissues. Therefore, more detailed observations focused on the results obtained with the latter two denaturation methods. MAP 2 immunoreactivity did not change in intensity and distribution over PMIs of 0.5-24 h (excluding the time required for the diffusion of fixatives into tissue, or for microwave irradiation), and the re- sponse of these tissues to enzymatic dephosphorylation also did not change during this time. How- ever, MAP 2 staining was more intense in sections cut from microwave-denatured tissue, and intersubject staining of MAP 2 antigens was more consistent in human tissues denatured by microwave energy. Intersubject variation in MAP 2 staining did not correlate with the PMI, the age of the

subject or the presence or absence of Alzheimer pathology such as neuritic plaques and neurofibrillary tangles. Finally, the results in the vibra- tome sections of bovine hippocampus did not differ appreciably from those in the paraffin sections of tissues fixed in the same manner, but cytological details were resolved better in paraffin sections.

Specific observations Hippocampus. Consistent with reports of the

preferential expression of MAP 2 in perikarya and dendrites in hippocampus and other CNS regions of experimental animals (e.g. Bernhard and Matus, 1984; Caceres et al., 1984; Olmstead, 1986; Dotti et al., 1987; Matus, 1988) our anti-MAP 2 antibodies labeled the somatodendritic domain of neurons in bovine hippocampus. Fig. 2 shows sections through 3 different regions of microwave-denatured bovine hippocampus (PMI = 12 h)probed with MAbs specific for MAP 2. Note that nearly all

Fig. 3. Microwave (A,B,D,F)-denatured and Bouin's (C,E) fixed postmortem hippocampus from a normal 85-year-old (A,B,D,F) and a 71-year-old subject with both Alzheimer's and Parkinson's disease (C,E). The sections were probed with anti-MAP 2 MAbs (M12 in C and E; T37 in A,B,D, and F). The panels show subiculum (A), CA3-CA4/dentate (B,C), CA4/dentate (D), presubioalum (E) and CA3 (F). Perikarya and proximal dendrites throughout the hippocatmpal formation contain intense MAP 2 imm,moreactivity, and MAP 2 staining of the neuropi] (asterisks in the stratum moleculare in B and D) also is seen. In contrast, white matter axons and gila (upper left area in F, upper fight area in C) are negative for MAP 2. A, D a n d F are at the same masnification (scale bar in A -- 100

pm) as are B, C and E (scale bar in C ffi 100 pro). Counterstained with hematoxylin.

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]76

neuronal perikarya and proximal dendrites are intensely MAP 2 positive in subiculum-CA1 (Fig. 2A), CA2 (Fig. 2B) and the dentate gyrus (Fig. 2C) whereas axons (e.g. those traversing CA2 in Fig. 2B) are negative.

The MAP 2 MAbs did not stain normal human hippocampal structures in formalin fixed brain, as reported by Kosik et al. (1984), while Bouin's fixed and microwave-denatured human hippocampus exhibited patterns of MAP 2 staining that were nearly the same as those seen in similarly treated bovine samples. Fig. 3 shows that MAP 2- specific MAbs stained the somatodendritic domain of human hippocampal neurons in both microwave-denatured (Fig. 3A, B,D,F) and Bouin's fixed (Fig. 3C,E) samples. These MAbs also stained thin processes (presumably distal dendrit ic branches) in the neuropil (e.g. in the stratum moleculare of the dentate in 3B,D), but they failed to stain white matter axons such as those in the alveus (3C,F). The ant i-MAP 2 MAbs did not stain tangles or other Ahheimer pathology as do many anti-tan (Grtmdke-Iqbal , 1986; Kosik et al., 1986; 1988) and some ant i -NF MAbs (Sternberger and Sternberger, 1985; Lee et al., 1988a, b).

Human neocortex, cerebellum, dorsal root ganglion, spinal cord, and intestine. In neocortex, cerebellum, dorsal root ganglion, spinal cord and the enteric nervous system of the intestine, MAP 2 immtmoreactivity was present in small processes, dendrites and neuronal perikarya of neocortex, cerebellum, dorsal root ganglion and the enteric nervous system (data not shown). Fig. 4 shows MAP2-positive neurons and dendrites in the posterior (Fig. 4A) and anterior (Fig. 4B) horn of spinal cord denatured by microwave treatment

Fig. 4. Sections of microwave-denatured (A,B) and Bouin's- fixed (C) normal postmortem human spinal cord obtained from the same case and probed with anti-MAP 2 (MS) MAbs. Note that M5 stains perikarya and proximal dendrites intensely in the posterior (A, insert in A) and anterior (13) horn of the microwave-denatured cervical cord while adjacent white matter is negative. In contrast, a section from an adjacent block of cervical cord fixed with Bouin's solution (C) shows negligible or no staining of the anterior horn. A-C are at the same magnification. The scale bar in the inset in A ffi 10 ~m, and the scale bar in Bffi100 ~m. Counterstained with hema-

toxylin.

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Fig. 5. Representative immunosta ined sections of normal pos tmortem h u m a n brain tissue to compare the immunoreactivi ty of N F proteins ( A - D ) and G F A P (E,F) in Bouin's (A,C,E) fixed and microwave (B,D,E)-denatured tissues in paraffin sections. The regions shown here include cerebellum ( A - D ) and entorhinal cortex (E,F). The sections were probed with MAbs to an epitope in N F - H that requires enzymatic dephosphorylat ion for recognition to occur (RM0304 in A and B), a phosphate- independent epitope in NF-L (RMS12 in C and D) or to G F protein (2.2B10 in E and F) as described elsewhere (Lee et al., 1984, 1986, 1987; Schmidt et al., 1987). Note that RMS12 and RM0304 are negative in Bouin's fixed tissue (A,C), but are intensely positive in microwave-denatured tissue ( B , D ) . G F immunoreactivi ty is seen in the Bouin 's fixed material (E), but it is more intense and cortical astrocytes are labeled to a greater extent in the microwave-denatured sample (F). A - D axe at the same magnification and the scale bar in A = 50 pm. E and F

are at the same magnification and the scale bar in E = 1 0 0 pm. Counterstained with hematoxylin.

178

while only weak MAP 2 staining was seen in gray matter of Bouin's fixed spinal cord (Fig. 4C).

N F and GF immunoreactivity in microwave-denatured tissues. Fig. 5 demonstrates the enhanced staining achieved in paraffin sections of microwave-denatured human tissue as compared with Bouin's solution using two anti-NF MAbs (Fig. 5A-D), and the MAb (2.2B10) to GF protein (Fig. 5E,F). One of these anti-NF MAbs, RMS12 (Fig. 5C,D), is specific for a core determinant in NF-L, while the other, RMS304 (Fig. 5D,E), is specific for a P ( - ) determinant in NF-H (Schmidt et al., 1987).

Studies of M A P 2 in human neural cell lines. None of the cell lines studied here exhibited MAP 2 immunoreactivity although some of the cell lines (D283, D341) previously were shown to express N F triplet proteins (He et al., 1989). Since cultured normal neurons have been shown to express MAP 2 (Chamak et al., 1987; Kosik and Finch, 1987; Dotti et al., 1987), it is possible that neop- lastic human cell lines regulate the expression of N F triplet proteins independently from the expression of MAP2.

Discussion

We have characterized for the first time the postmortem stability and normal distribution of immunoreactive MAP 2 ill human hippocampus, and in other regions of the human CNS and PNS. The demonstration of human MAP 2 in situ, was made possible by using Bouin's solution and espe- cially microwave energy to denature brain tissue as well as a new library of anti-MAP 2 MAbs. The relative segregation of MAP2 immunoreactivity into perikaryal and dendritic domains of postmortem human hippocampal neurons is similar to that described in experimental animals (reviewed in Olmstead, 1986; Matus, 1988), and to our bovine data. However, we could not confirm that MAP2 is expressed in astrocytes (Papasozomenos and Binder, 1986). This could reflect heterogeneity among MAP 2 in different species or brain regions due to post-translational modifications, such as phosphorylation, or other factors (Kosik et al., 1984; Fischer et al., 1987; Matus, 1988).

Microwave denaturation of tissue has been shown to have several advantages over the use of chemical fixatives alone (Merrit and Frazer, 1977: Marani et al. 1987). For example, in addition to effectively preserving the immunological properties of MAP 2, N F and GF in situ, microwaves shorten 'fixation' times to minutes while chemical fixatives require hours to penetrate and fix tissues. Also, compared with frozen sections, microwave- denatured tissues stained better with our MAbs and retained cytological details more effectively (Schmidt et al., unpublished). Although the pre- cise mechanism whereby microwaves denature proteins is unknown, MAP 2 is a heat-stable MAP that may retain its immunological properties to a greater extent in microwave-treated tissues. Re- gardless of the underlying mechanism of antigen preservation, we anticipate that the denaturation of postmortem brain tissue with microwaves will prove valuable in efforts to probe the proteases, kinases, phosphatases and other potentially labile enzymes which regulate the metabolism of MAP 2.

Acknowledgements

This work was supported by National Institute of Health Grants AG-06107. AG-06901, MH- 43880, CA-36245 (Merit Award), NS-18616 (Sen. Jacob Javits Award), and AG-06901. Appreciation is expressed to Mr. P. Newman and C. Page for technical assistance, to Drs. H. Friedman and D.D. Bigner for access to the cell lines, and to Dr. R. Gur, Dr. G.P. Pietra, Dr. L.B. Rorke, and Mr. J. DiRienzi for help with tissue collection. Ms. H. Comstock of the Philadelphia Alzheimer's Disease And Related Disorder Association, Inc., Dr. M. Kurtz and associates at the Elwyn Institutes, and the families of the patients studied here made our work possible through their generous efforts to foster research.

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Distribution of phosphate-independent MAP2 epitopes revealed with monoclonal antibodies in microwave-denatured human nervous system tissues