Perikaryal accumulation and proteolysis of neurofilament proteins in the post-mortem rat brain

Neurobiotogy of Aging, Vol. 16, No. 4, pp. 651-660, 1995 Copyright © 1995ElsevierScienceLtd. Printed in the USA. All rights reserved 0197-4580/95 $9.50 + .00

Pergamon 0197-4580(95)00062-3

Perikaryal Accumulation and Proteolysis of Neurofilament Proteins in the Post-Mortem Rat Brain J A M E S W. G E D D E S , V I M A L A B O N D A D A , T I N A L. T E K I R I A N , Z H E N P A N G A N D R O B E R T G. S I M A N *

Sanders.-Brown Center on Aging and Department o f Anatomy and Neurobiology, University o f Kentucky, Lexington, K Y 40536-0230 *Cephalon Inc., West Chester, PA 19380 R e c e i v e d 1 N o v e m b e r 1994; Rev i sed 20 D e c e m b e r 1994; A c c e p t e d 16 J a n u a r y 1995 GEDDES, J. W., V. BONDADA, T. L. TEKIRIAN, Z. PANG AND R. G. SIMAN. Perikaryalaccumulation andproteolysis of neurofilamentproteins in the post-mortem rat brain. NEUROBIOL AGING 16(4) 651-660, 1995.-Investigations of neurofilament alterations in neurodegenerative disorders utilize postmortem human tissues obtained at autopsy. To determine if alterations in the levels or distribution of neurofilament proteins might occur during the interval between death and autopsy, the postmortem cooling curve of the human brain was modeled in Sprague-Dawley rats and neurofilament proteins were examined by immunocytochemistry and immunoblots. One hour after death, enhanced perikaryal immunostaining of NF-M and both phosphorylated and nonphosphorylated NF-H epitopes was observed throughout the hippocampal formation. A greater number of neurons exhibited increased somatic immunostaining 4-h postmortem. In addition, loss of neurofilament protein immunostaining was observed in the neuropil, particularly in the molecular layer of the dentate gyrus. This corresponded with, but lagged behind, the pattern of calpain activation determined using an antibody against calpain-cleaved a-spectrin. Immunoblots confirmed the postmortem loss of neurofilament proteins in both triton-soluble and insoluble fractions. These results demonstrate that the levels and localization of neurofilament proteins observed in tissues obtained at autopsy even with short postmortem intervals may not accurately reflect the premortem condition. Aging

Calpain

Hippocampus

Neurofilaments

Postmortem

Rat

Sprague-Dawley

Investigation of neurofilament alterations in neurodegenerative disorders involves the use of postmortem tissues obtained at autopsy. Although it has been suggested that there is not a correlation between postmortem delay and the presence of neurofilament epitopes (6), this has not been examined directly. Postmortem delay affects the levels, localization, and phosphorylation of microtubule-associated proteins (33). In addition, degradation of neurofilament proteins is evident following cerebral ischemia (15,25,28), suggesting that similar alterations may occur postmortem. Neurofilaments are composed of three proteins with apparent molecular weights of 200, 160, and 68 kDa, referred to as neurofilament heavy (NF-H), medium (NF-M), and light (NF-L), respectively. The neurofilament proteins are phosphorylated in vivo, particularly at multiple repeats of the Lys-Ser-Pro (KSP) motif (19,20). Neurofilament proteins are good substrates for

AN altered distribution of neurofilament proteins is observed in several neurodegenerative disorders. In the normal mammalian central nervous system (CNS), highly phosphorylated neurofilament proteins are present in axons whereas those in the cell bodies and dendrites react poorly with antibodies against phosphorylated neurofilaments (34,36). In amyotrophic lateral sclerosis (22), Parkinson's ,disease (8,9), and several cerebellar disorders (35), phosphorylated neurofilaments accumulate in neuronal perikarya. In Alzheimer's disease (AD), many antibodies against phosphorylated neurofilaments label neurofibrillary tangles (1,7), although much of this is due to cross-reaction with abnormally phosphorylated tan (16,27). However, tangles are also immunostained by anti-neurofilament antibodies which do not cross react with hyperphosphorylated tan (18,38). In addition, nonphosphorylated neurofilament epitopes are lost from tangle-bearing neurons (24).

l Requests for reprints should be addressed to James W. Geddes, Ph.D., 209 Sanders-Brown Building, University of Kentucky, Lexington, KY 40536-0230. 651

652

GEDDES ET AL.

calcium activated proteases (calpains) (21,26,32) and their susceptibility is enhanced by dephosphorylation (29). The purpose of this study was to determine if alterations in the levels, localization, or phosphorylation of neurofilament proteins occur during postmortem intervals associated with obtaining human brain tissue at autopsy. METHOD

Postmortem Model Male Sprague-Dawley rats, approximately 3 months old and weighing 250-300 g, were killed using sodium pentobarbital (100 m g / k g ) o r t00°70 carbon dioxide. The postmortem cooling curve of the human brain was simulated as described previously (10,33). After postmortem intervals of 1 h to 8 h, the brain was removed and placed in 4°7o paraformaldehyde, 4°C. Control animals were anesthetized with sodium pentobarbital (50 mg/kg) perfused transcardially with 100 ml 0.9% saline followed by 300 ml of 4°7o paraformaldehyde in 0.1 M Soerensen phosphate buffer. All brains were postfixed for 24 h in the same fixative and cryoprotected in 30% sucrose prior to freezing in powdered dry ice. The frozen tissues were stored at -70°C. All results were replicated in a minimum of three animals. For the immunoblot experiments, animals were killed with sodium pentobarbital or carbon dioxide as described above. The human postmortem cooling curve was simulated and at the appropriate postmortem interval (0-8 h after death), the brains were removed, frozen in powdered dry ice, and stored at - 7 0 ° C .

Immunohistochemistry Sections (30 t~m) were cut in coronal and horizontal planes with a freezing microtome. Free floating tissue sections were rinsed 3 times in Tris buffered saline (TBS, 50 mM, pH 7.5). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in TBS for 15 min. Sections were transferred into TBS containing 0.1 O7oTriton X-100 (TrisA), followed by incubation in TrisA containing 1.5 °7o normal horse serum. The primary antibodies used in the immunohistochemistry included anti-NF-L (Sigma, clone NR4, mouse IgGl), anti-NF-M (Sigma, clone NN19, mouse IgG1), and anti-NF-H (ICN, clone NE14; Amersham, clone N52; Sternberger Monoclonals, clones SMI-31 and SMI-32, both are mouse IgG1). Sections were also immunostained using antibodies against microtubule-associated proteins tau and MAP2 (Tau-1 and API4, respectively), and using A/3-37, an antibody directed against a short peptide corresponding to the COOH-terminus of the NH2 terminal of a fragment

(CQQQEVY) of the c~-subunit of brain spectrin produced by calpain-I mediated proteolysis. It was raised against the same peptide and is very similar to the A/3-38 antibody characterized previously (29). This antibody does not react with intact spectin nor with a-spectrin fragments produced by several other mammalian proteases. The characterization of each of the antibodies used in this study is summarized in Table I. Tissue sections were incubated overnight at room temperature with the primary antibody. Following three rinses in Tris A, immunostaining was performed using a biotinylated anti-mouse (or anti-rabbit for A/3-37) secondary antibody preadsorbed against rat serum and the avidin-biotin technique (Elite ABC kit, Vector Laboratories). Diaminobenzidine was used as the peroxidase substrate. Corresponding sections were stained with Cresyl violet. The specificity of antibodies was confirmed using immunoblots and controls for the specificity of the immunohistochemistry involved omission of the primary antibody and immunostaining with mouse IgG (Vector). Some sections were treated with alkaline phosphatase (Sigma type VII-S, 100 U/ml) for 2 h at 37°C in Tris buffered saline (pH 8.3) prior to incubation with the primary antibody.

SDS Gel Electrophoresis and Immunoblotting The frozen brains were homogenized (20°70 w/v) in TBS containing protease inhibitors: 100 #M leupeptin (BoehringerMannheim), 1 #M pepstatin (Boehringer-Mannheim), 1 mM ethylenediamine-tetraacetic acid (EDTA, Sigma) and 200/~M 4-(2-aminoethyl)-benzenesulfonyl fluoride, HCL (AEBSF, Calbiochem) and 0.5 o70 Triton-X- 100 (v/v). Following centrifugation at 100,000 x g for 30 min at 4°C, the supernatant was collected and the Triton-insoluble pellet was resuspended in the homogenization buffer. Protein content was determined using the BCA protein assay (Pierce) and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (17) using a mini-gel apparatus (Bio-Rad). Samples of supernatant and pellet containing 10-20/zg protein were electrophoresed in gels of 6.5% polyacrylamide (Bio-Rad). Protein standards of known molecular mass were also run on each gel. Following SDS-PAGE, proteins were electrophoretically transferred onto nitrocellulose using a mini trans-blot apparatus (Bio-Rad). Nonspecific binding was blocked with 1.5°70 normal horse serum in Tris-buffered saline for 1 h and the blots were incubated with primary antibodies overnight at room temperature. The blots were rinsed in Tris-Tween buffer (10 mM Tris, 15 mM NaC1 and 0.507o Tween 20), incubated with alkaline-phosphatase anti-mouse IgG or anti-rabbit IgG (Sigma),

TABLE 1 C H A R A C T E R I Z A T I O N OF ANTIBODIES USED IN THIS STUDY Clone

NE 14 SMI 31 NN 19 N52 SMI-32 NR4 AP 14 Tau-I Ab-37

Antibody Against:

References

Phosphorylated NF-H Phosphorylated NF-H NF-M Non-phosphorylated NF-H Non-phosphorylated NF-H NF-L MAP2a,b non-phosphorylated tau c~-spectrin breakdown products

(34) (36) (34) (34) (36) (34) (3,5) (4,30,37) (31)

Comments

May also recognize MAP2 (27) Also recognized NF-M on immunoblots

Does not recognize intact c~-spectrin

POSTMORTEM ALTERATIONS IN NEUROFILAMENT PROTEINS rinsed and developed using BCIP/NBT (Promega). Control blots were similarly treated, except that the primary antibody was omitted. Densitometric analysis was used for relative quantitation of the Western blots. The blots were digitized using a Macintosh IIci computer (Apple) equipped with an image capture card (Data Translations), an Hitachi CCD camera and Nikon macro lens. The digitized images were evaluated using the NIH Image software (v 1.55) which includes a subroutine for the analysis of gels and blots. Differences between groups were determined using two-way analysis of variance (ANOVA), and post hoc comparisons were performed using Student's t test. RESULTS Analysis of neurofilament immunocytochemistry focused on the hippocampal formation. The anatomical organization of this region is well characterized and a previous study examined postmortem alterations in micrombule-assodated proteins (MAP 1b, MAP2, tau) in the hippocampai formation (33). Results obtained with rats killed using sodium pentobarbital versus carbon dioxide were indistinguishable, demonstrating that the anesthetic did not significantly influence the postmortem alterations in neurofilament proteins. In the perfused rat brain, antibody NN-19 (anti NF-M) intensely labeled mossy fiber axons along with axons in the inner

653

molecular layer of the dentate gyrus (Fig. 1 and Fig. 2). Moderate staining was observed in the outer 2/3 of the dentate gyrus molecular layer and across the hippocampal fissure in stratum lacunosum-moleculare. A slightly intensified band of immunostaining was evident in the pyramidal cell layer of both CAI and CA3. Axons in the alveus and corpus callosum were strongly labeled. Staining was light in other hippocampal regions examined. Antibodies against phosphorylated NF-H (SMI 31 and NE 14) produced a similar pattern of immunostaining except that the mossy fiber axons were lightly immunostained (Fig. 3). In contrast, antibodies against nonphosphorylated neurofilament epitopes (N52, SMI-32) immunostained occasional dendrites throughout the hippocampai formation. Neuropil staining was also observed, which was most intense in stratum radiatum and stratum oriens of CA3 but light in CA1 and in the dentate gyrus molecular layer. In the occasional neuron, the perikarya was lightly immunostained (Fig. 4). The anti-NF-L antibody used in this study (NR4) does not work well for immunocytochemistry in paraformaldehyde fixed tissues. One hour after death, perikaryal immunostaining was observed with each of the anti-NF antibodies. This was evident in select neurons in each hippocampal region but was most prominent in CAI s. oriens. A greater number of neurons exhibited enhanced perikaryal immunostaining4 h and 8 h after death (Fig. 3). At these later time points, loss of NF-M immunostaining in the outer 2/3 of the dentate gyrus molecular layer was

FIG. 1. NN 19 (NF-M) immunoreactivity in the rat hippocampal formation. In a rat perfused transcardially with 4°70paraformaldehyde (A), NFM immunostaining is prominent in mossy fiber axons and in the inner molecular layer of the dentate gyrus but is not observed in pyramidal neurons or in granule cells of the dentate gyrus. A similar pattern of immunostaining is observed 1 h postmortem (B). By 4 h postmortem (C), loss of immunostaining is evident in the outer molecular layer of the dentate gyrus (indicated by large open arrowhead). In addition, perikaryal immunoreactivity is evident in some pyramidal neurons (indicated by small black arrow). The pattern of immunostaining observed 8 h after death (D) is similar to that observed in 4 h postmortem animals. The region enclosed by the box in (A) is shown at higher magnification in Fig. 2. Abbreviations: dg, dentate gyrus; mf, mossy fibers. Scale bar = 1 ram.

654

G E D D E S ET AL.

FIG. 2. NN 19 (NF-M) immunoreactivity in the dentate gyrus and CAI of the rat hippocampal formation. The postmortem alterations in NF-M immunostaining are shown in higher magnification as compared to Fig. 1. Note the loss in immunostaining in the outer molecular layer of the dentate gyrus in the 4 h (C) and 8 h postmortem (D) rats as compared to control (A) and 1 h postmortem animals (B). Abbreviations: g, granule cells of the dentate gyrus; iml, inner molecular layer of the dentate gyrus; Im, stratum lacunosum-moleculare of CA1; oml, dentate gyrus outer molecular layer; or, CA1 stratum oriens; rad, CAI stratum radiatum; pyr, CAI stratum pyramidale. Scale Bar (D) = 200 #m.

observed, as well as C A I s. lacunosum-moleculare (Fig. 1). With antibodies against nonphosphorylated neurofilament epitopes, loss of neuropil staining was evident in CA1 s. radiatum and s. oriens, and in the C A 3 / h i l a r region (Fig. 3). Immunostaining with antibodies against phosphorylated N F - H (SMI-31 and NE 14) was intensified in a band surrounding the CA1 and CA3 pyramidal ceils and in the innermost portion of the dentate gyrus molecular layer, but was decreased in other regions including CA1 s. radiatum and s. oriens (Fig. 3). Mossy fiber staining progressively diminished during the 8-h postmortem interval examined in this study. Additional sections were immunostained with antibodies against microtubule-associated proteins, tau (tau-l) and M A P 2 (AP-14), and against an c~-spectrin-breakdown product resulting from calpain proteolysis (Aj3-37). For tan and M A P 2 , the postmortem alterations were similar to those described previously (33). These differ from those observed with anti-NF antibodies in that enhanced somatic immunoreactivity is observed in all pyramidal and granule cell neurons throughout the hippocampal formation in contrast to the select neurons exhibiting enhanced perikaryal N F immunostaining (Fig. 3). The spatial and temporal pattern of calpain activation, indicated by A/3-37 immunostaining, preceded the perikaryal accumulation and loss of neurofilament proteins and coincided with the loss of MAP2. One hour after death, perikaryal accumulation of spectrin breakdown products is observed in select neurons throughout the hippocampal formation. By 4 h postmortem, immunostaining of spectrin breakdown products is prominent in dendrites o f dentate gyrus granule cells and also evident in C A I but not in CA3 or hilus (Fig. 5). A loss of neurofilament proteins was also evident with immunoblots. One hour after death, the levels o f neurofilament proteins were not significantly different from control levels (results not shown). Four hours postmortem, a loss o f neurofilament proteins was evident in both the triton-insoluble pellet and supernatant fractions (Table 2, Fig. 6 and Fig. 7) with the decrease

being more pronounced in the supernatant. Breakdown products of N F - L were observed with both the NR4 and NE14 antibodies. The A/~-37 a n t i b o d y detected spectrin b r e a k d o w n products which increased during the postmortem interval but were undetectable in the control (0 h postmortem) rat brain homogenates (results not shown). DISCUSSION The results of this study demonstrate that the levels and localization of neurofilament proteins are altered in the postmortem rat brain under conditions which simulate the cooling rate of the human brain after death. In animals transcardially perfused with fixative, phosphorylated neurofilament epitopes were not observed in neuronal neuronal perikarya. Nonphosphorylated neurofilaments were present to a limited extent in the soma of some neurons. Within 1 h after death, enhanced perikaryal im-

TABLE 2 RELATIVE POSTMORTEM NEUROFILAMENTPROTEIN LEVELS DETERMINED FROM IMMUNOBLOTS Triton-Insoluble P e l l e t

Triton-Soluble Supernatant

Antibody

Control

4 h Postmortem

Control

4 h Postmortem

NE 14 N52 SMI-31 SMI-32 NN 19 NR4 AP14 Tau-1

100 + 9 100 ± 9 100 _+ 2 100 ± 15 100 ± 10 100±13 100 _-z-8 100_ 7

54 _+ 13"* 66 + 6** 65 + 12" 54 ± 15" 30 ± 5** 27_+1"* 73 ± 18 100 ± 21

100 +_ 7 100 _+ 8 100 ± 8 100 _ 5 100 ± 10 100+15 100 ___5 100 + 9

29 +_ 13"* 26 +_ 7** 40 ± 4** ND** 30 ± 5** 11+6"* 44 ± 4** 101 ± 1

Values are expressed as percent of control values and are the mean +_SD, n -- 3. Significance: *p < 0.05, **p < 0.01; ND = not deleted.

Ji

:IG. 3. Immunostaining in the dentate gyrus and CA1 of the rat hippocampal formation in control and 4 h postmortem animals. (A) SMI-31 (phosphorylated NF-H) immunoreactivity in Lcontrol rat. (B) Four hours postmortem, SMI-31 immunoreactivity is intensified in a band near the pyramidal neurons and in the innermost region of the dentate gyrus molecular layer. ;taining intensity was decreased in many other regions of the hippocampal formation such as CA1 s. oriens and radiatum. Intense perikaryal immunoreactivity is also evident in some pyramilal and granule cell neurons. (C) SMI-32 immunoreactivity (nonphosphorylated NF-H) in a control rat. Immunostaining is evident in the neuropil and occasional dendrite whereas neuronal :ell bodies are largely unstained. (D) Four hours postmortem, many neurons exhibit perikaryal SMI-32 immunoreactivity and dendritic staining is intensified. In contrast, the neuropil stainng is diminished as compared to that observed in transcardially perfused (0 h postmortem) rats. Tau-I immunostaining in a control (E) and 4 h postmortem rat (F). During the postmortem nterval, immunostaining shifts from the neuropil to neuronal perikarya. AP-14 (MAP2) immunostaining in a control (G) and 4 h postmortem (H) also exhibits perikaryal accumulation durng the postmortem interval. Intensification of immunostaining in proximal dendrites is also observed and immunoreactivity is diminished in the outer molecular layer of the dentate gyrus. ~,bbreviations are the same as for Fig. 2. Scale bar (H) = 200 #m.

656

G E D D E S ET AL.

FIG. 4. Perikaryal accumulation of NF immunoreactivity. In CA1 s. oriens of a control rat (A), SM1-32 is evident in dendrites. In an occasional cell body, light immunoreactivity is observed in the cytoplasm. Four hours postmortem (B), many neurons exhibit intense perikaryal SMI-32 immunoreactivity. NE 14 immunoreactivity is punctate in CA1 s. oriens of control animals (C) but is absent from neuronal cell bodies. Four hours postmortem (D), intense immunoreactivity is observed in the somatodendritic compartment of many neurons. Scale bar (D) = 50/~m.

munoreactivity o f b o t h phosphorylated a n d n o n p h o s p h o r y l a t e d n e u r o f i l a m e n t epitopes was evident in select neurons. This persisted t h r o u g h o u t the 8 h p o s t m o r t e m interval e x a m i n e d in this study. In addition, at 4 h a n d 8 h p o s t m o r t e m , loss o f neurofil-

a m e n t proteins was evident in b o t h the triton-soluble a n d insoluble fractions. These results suggest that alterations in the levels a n d localization of neurofilarnent proteins observed in postmortem h u m a n tissues m u s t be interpreted with caution.

P O S T M O R T E M A L T E R A T I O N S IN N E U R O F I L A M E N T

PROTEINS

.... ii

FIG. 5. AB-37 immunostalning in the rat hippocampal formation at 0, 1 h, and 4 h postmortem. This antibody detects spectrin breakdown products resulting from calpain activation. In an rat transcardially perfused with 4070paraformaldehyde (A), very faint immunostaining is observed throughout the hippocampal formation. One hour after death (B), spectrin breakdown products fill many neurons throughout the hippocampal formation. In addition, the intensified immunostaining is observed in the dentate gyrus and across the hippocampal fissure stratum lacunosum-moleculare. Four hours postmortem (C), AB-37 immunostalning is also evident in dendrites of CA1 but not CA3 pyramidal neurons. The perikaryal staining of individual neurons is largely absent at this time point. Scale bar (C) = I mm.

657

658

Oh

4h

Oh

NE14

4h

SMI31

SMI-32 N52 NN19

o AP14

NR4

Tau-1 i

~ , 1 1 1 1 1 1 1 1 1 1 m ¸~

I

FIG. 6. Immunoblots of neurofilament proteins in the triton-insoluble pellet obtained from control (0 h) and 4 h postmortem rat brain. The individual lanes represent separate animals. Each antibody revealed a loss of neurofilament protein during the 4 h postmortem interval. The immunoblots also demonstrate that the NN 19 antibody detects both NF-M and NF-L, and neurofilament breakdown products are observed with this antibody and with NR4 in the postmortem rats. Quantitation of the immunoblots is indicated in Table 2.

The altered neurofilament immunoreactivity likely results from a shift in the cellular localization of neurofilament proteins, although it is also possible that it reflects changes in the antigenicity of neurofilament epitopes in different neuronal compartments. For example, epitopes could be unmasked by changes in conformation or proteolysis after death. However, enhanced perikaryal immunostaining was observed with antibodies against both phosphorylated and nonphosphorylated epitopes of NF-H and NF-M and also against other cytoskeletal proteins including tau and MAP2. In each case, the increase in somatic immunoreactivitywas accompanied by decreased immunostaining in neuronal processes. Neurofilament proteins are excellent substrates for calpains (calcium-activated neutral proteases) (21,26,32) and several observations provide support for the involvement of calpains in the postmortem proteolysis of neurofilament proteins. The appearance of breakdown products with NF-L resembles that produced by calpain proteolysis (12). The perikaryal accumulation of neurofilament proteins during the postmortem interval is similar to that observed for spectrin breakdown products resulting from calpain proteolysis, except that neurofilament proteins persisted in the soma throughout the 8 h time period examined. The loss of immunostaining for NF-M in the dentate gyrus molecular layer at 4 h postmortem also corresponds to a

FIG. 7. Immunoblotsof neurofilamentand microtubule-associatedproteins in the supernatant fraction obtained from 0 h and 4 h postmortem rat brain. Each lane represents a separate animal. Loss of both phosphorylated (SMI-31) and nonphosphorylated (N52) NF-H was observed during the 4 h postmortem interval. The levelsof other neurofilament proteins also decreased (Table 2). A similar loss of MAP2 (AP14) was observed. The shift in electrophoretic migration of tau-I immunoreactivebands reflects dephosphorylationof tau during the postmortem interval.

region of high calpain activity. In addition, the time course of neurofilament degradation on immunoblots approximates but lightly lags behind, the temporal profile of calpain activation. The possible involvement of additional proteases is suggested by the multiple breakdown products of NF-L, in contrast to the single band observed following incubation of neurofilament proteins with calpain in vitro (12). The neurofilament proteins differed in their sensitivity to postmortem alterations. NF-L was extremely sensitive to proteolysis whereas NF-H was most resistant. This is consistent with the observation that calmodulin binds to NF-H but not NF-M or NF-L and protects it from calpain (12). The extensive phosphorylation of NF-H, as compared to NF-M and NF-L, may also protect it from calpain mediated proteolysis (21) and noncalcium dependent proteases (11). The triton-soluble neurofilaments were more vulnerable to proteolysis than those in the insoluble pellet. The reason for this difference is unclear but may reflect differences in the accessibility to proteases of neurofilament proteins in the two fractions. Although tau can be degraded by calpain, a greater enzymesubstrate ratio is required as compared to MAP2, which is a very sensitive calpain substrate (13,14). The apparent sparing of tau relative to MAP2 and neurofilament proteins also reflects enhanced tau-1 immunoreactivity in the postmortem rat brain resulting from tan dephosphorylation and the phosphorylationsensitive nature of the tan-1 epitope (30,37). In the normal adult brain, tau is more extensively phosphorylated in the somatoden-

P O S T M O R T E M A L T E R A T I O N S IN N E U R O F I L A M E N T P R O T E I N S dritic versus axonal compartment (30) whereas the reverse is true for neurofilament proteins. Increased abundance of dephosphorylated neurofilament epitopes was not observed in the postm o r t e m rat brain, in contrast to the dephosphorylation o f tau. Activation of phosphatases in the somatodendritic compartment could result in the dephosphorylation o f tau but not NFs. A second possibility is that because phosphorylation protects NFs from proteolysis, dephosphorylation may result in rapid proteolysis and prevent the accumulation of nonphosphorylated neurofilament proteins (see 11). The postmortem increase in calpain activation in the dentate gyrus molecular layer and CA1 corresponds to the distribution of N-methyl-D-aspartate (NMDA) receptors (23). Activation of the N M D A receptors would be expected to occur postmortem both due to an elevation in extracellular glutamate as in ischemia (2) and to metabolic inhibition, which can result in activation of the N M D A receptors in the absence of an elevation in extracellular glutamate (39). The lack of calpain activation in CA3 in the postmortem rat brain is not due to the absence o f the protease, as kainic acid injection results in calpain activation and loss of M A P 2 in CA3 pyramidal neurons (results not shown).

659

The mechanisms which lead to the perikaryal accumulation o f neurofilament proteins are uncertain. It may reflect calpain activation and the accumulation o f neurofilament breakdown products. Alternatively, the observation that both axonal and dendritic microtubule-associated proteins also accumulate in neuronal perikarya in the postmortem rat brain (33) suggests a general collapse of the cytoskeleton. In any event, the fact that perikaryal accumulation of both phosphorylated and nonphosphorylated neurofilament epitopes occur~ during the postmortem interval suggests caution in the interpretation of results obtained with human brain tissues obtained at autopsy. In these tissues, increased perikaryai immunostaining could reflect alterations occurring during the course of the disease but might also result from changes occurring in the premortem agonal state or postmortem interval. ACKNOWLEDGEMENTS We thank L. I. (Skip) Binder, Molecular Geriatrics Corp., for the generous gift of tau-I and API4 antisera, and Gall Johnson (University of Alabama at Birmingham) for helpful discussion. This work was supported by NIA Grant AG10678 to J.W.G.

REFERENCES 1. Anderton, B. H.; Breinburg, D.; Downes, M. J.; Green, P. J.; Tomlinson, B. E.; Ulrich, J.; Wood, J. N.; Kahn, J. Monoclonal anti-/ bodies show that neurofibriillary tangles and neurofilaments shar~ antigenic determinants. Nature 298:84-86; 1982. 2. Benveniste, H. The excitotoxin hypothesis in relation to cerebral ischemia. Cerebrovascular and brain metabolism reviews. 3:213/245; 1991. 3. Binder, L. I.; Frankfurter, A.; Kim, H.; Caceres, A.; Payne, M. R.; Rebhun, L. I. Heterogeneity of microtubule-associated proteir~ 2 during rat brain development. Proc. Natl. Acad. Sci. USA. 81:56135617; 1984. 4. Binder, L. I.; Frankfurter, A.; Rebhun, L. I. The distribution!of tau in the mammalian cen~Lral nervous system. J. Cell Biol. 101: 1371-1378; 1985. 5. Binder, L. I.; Frankfurter, A.; Rebhun, L. I. Differential localization of MAP-2 and tau in mammalian neurons in situ. Ann NY Acad Sci. 466:145-166; 1986. 6. Blanchard, B. J.; Ingram, V. M. Age-related neurofilament phosphorylation in normal human brains. Neurobiol. Aging 10:253258; 1989. 7. Dahl, D.; Selkoe, D. J.; Pero, R. T.; Bignami, A. Immunostaining of neurofibrillary tangles m Alzheimer's senile dementia with a neurofilament antiserum. J. Neurosci. 2:113-119; 1982. 8. Forno, L. S.; Sternberger, L. A.; Sternberger, N. H.; Strefling, A. M.; Swanson, K.; Eng, L. F. Reaction of Lewy bodies with antibodies to phosphorylated and nonphosphorylated neurofilaments. Neurosci. Lett. 64:253-258; 1986. 9. Galloway, P. G.; Grundke, I. I.; Iqbal, K.; Perry, G. Lewy bodies contain epitopes both shared and distinct from Alzheimer neurofibrillary tangles. J. Neuropathol. Exp. Neurol. 47:654-663; 1988. 10. Geddes, J. W.; Chang, C. H.; Cooper, S. M.; Lott, I. T.; Cotman, C. W. Density and distribution of NMDA receptors in the human hippocampus in Alzheimer':~ disease. Brain Res. 399:156-161; 1986. 11. Goldstein, M. E.; Sternberger, N. H.; Sternberger, L. A. Phosphorylation protects neurofilaments against proteolysis. J. Neuroimmunology 14:149-160; 1987. 12. Johnson, G. V. W.; Greenwood, J. A.; Costello, A. C.; Troncoso, J. C. The regulatory role of calmodulin in the proteolysis of individual neurofilament proteins by calpain. Neurochem. Res. 16:869873; 1991. 13. Johnson, G. V. W.; Jope, 1~',S.; Binder, L. I. Proteolysis oftau by calpaln. Biochem. Biophys. Res. Commun. 163:1505-1511; 1989. 14. Johnson, G. V. W.; Litersky, J. M.; Jope, R. S. Degradation of

15.

16.

17. 18.

19.

20.

21. 22.

23. 24.

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