Enhancement of immunoreactivity for NF-κB in the hippocampal formation and cerebral cortex of Alzheimer's disease

BRAIN RESEARCH ELSEVIER

Brain Research 735 (1996) 159-168

Short communication

Enhancement of immunoreactivity for NF-KB in the hippocampal formation and cerebral cortex of Alzheimer's disease Kazuhiro Terai, Akinori Matsuo, Patrick L. McGeer * Kinsmen Laboratory, of Neurological Research, The Uniuersity of British Columbia, 2255 Wesbrook Mall, Vancouuer B.C. V6T IZ3, Canada

Accepted 5 March 1996

Abstract

The distribution of nuclear factor-kappa B (NF-K B) was investigated immunohistochemically in the hippocampal formation, entorhinal cortex, middle temporal gyms and visual cortex of Alzheimer's disease (AD) and control postmortem cases using a polyclonal antibody against the NF-KB p65 subunit. In AD cases, prominent staining for NF-KB was seen in neurons and their processes, neurofibrillary tangles and dystrophic neurites. In control cases, only weak staining of some neurons was obtained. The neuronal staining observed in AD was strongest in the hippocampal formation and entorhinal cortex, less in the middle temporal gyms and least in the visual cortex. There was no difference between AD and control cases in the staining of glial cells and vascular walls. These results suggest that enhanced expression of neuronal NF-KB occurs in areas affected by AD pathology. Keywords: Immunohistochemistry; Nuclear factor-KB; Alzheimer's disease; Human brain; Neuron; Hippocampal formation; Cerebral cortex

Nuclear factor-kappa B (NF-KB) was identified in 1986 as a gene expression enhancer for immunoglobulin K light chains in B-lymphocytes [32]. Initially, it was believed that the molecule was restricted to B-lymphocytes and appeared only at an appropriate stage of maturation. Many studies since then have demonstrated that NF-KB exists in cells of diverse lineage and that it can be induced by many molecules [6,33] including inflammatory mediators such as lipopolysaccharides [31] and cytokines [23]. NF-KB induces transcription of immune-related molecules other than the K gene in B-lymphocytes, including cytokines [13,20], complement proteins [22,40], M H C class 1 and class 2 glycoproteins [2,3], [32-microglobulin [8] and others [21]. Presently NF-KB is regarded as a central regulator of inflammatory, immune and acute phase reactions. NF-KB is a heterodimer composed of p50 and p65 subunits. The p65 subunit has a nuclear translocation signal sequence which is masked by IKB, a specific inhibitor of NF-KB, which holds it in the cytosol [5,27]. Release is initiated when specific kinases, activated by cell surface receptors, phosphorylate IKB. Proteolysis of IKB then occurs, releasing the p65 NF-KB subunit which is

* Corresponding author. Fax: [email protected]

+ 1 (604) 822-7086; E-mail:

translocated to the nucleus. There it binds to a specific D N A sequence called the B motif [32], which is the gene expression enhancer. More details of the NF-KB system can be found in review articles [4,19]. The neuropathological features of A l z h e i m e r ' s disease (AD) are a widespread appearance of neurofibrillary tangles and [3-amyloid containing senile plaques. Evidence is accumulating that inflammatory components such as cytokines and complement proteins play a significant role in exacerbating neuronal damage in A D [14-16,28,29,34]. Recently, it has been reported that rat and mouse neurons have constitutive NF-KB activity [10,26,38] and that advanced glycation end products on neuronal paired helical filaments in A D brain may cause transcription activation via NF-KB [39]. However, localization of NF-KB has not yet been clarified in human brain. Therefore we have investigated NF-KB immunostaining in the hippocampal formation and cerebral cortex of A D and control cases. A rabbit polyclonal antibody to the NF-KB p65 subunit (diluted 1:300) was used in the present study. The antibody was raised against a synthesized peptide corresponding to amino acid residues 5 3 1 - 5 5 0 of the p65 subunit of human NF-KB (sc-372; Santa Cruz Biotechnology). The synthesized peptide (control peptide; Santa Cruz Biotechnology) was used for immunoabsorption and Western blot control experiments.

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0006-8993(96)00310- 1

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162

K. Terai et al. / Brain Research 735 (1996) 159 168

For Western blots, fresh tissues of human hippocampus were homogenized in five volumes of ice-cold 10 mM Tris-HC1 (pH 7.4) containing 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, phenylmethyl-sulfonyl-fluoride (100 p~g/ml), leupeptin (1 Ixg/ml), pepstatin (1 p~g/ml) and aprotinin (1 Ixg/ml). The homogenate was centrifuged at 15000 rpm for 20 min at 4°C. The supernatant was collected as a crude cytosol fraction• Fractions containing about 100 txg of protein were used for 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinyliden difluoride membrane (Immobilon-P, Millipore, Bedford). The membrane was blocked for 30 min with 5% skim milk in l0 mM Trisbuffered saline (pH 7.4) containing 0.1% Tween 20 (TBST) at room temperature and then incubated overnight with the primary antibody in TBS-T containing 1% skim milk. After washing with TBS-T, the membrane was reacted for 2 h at room temperature with alkaline phosphatase-labeled anti-rabbit IgG (1:5000, GIBCO BRL, USA) in TBS-T containing 1% skim milk. The labeling was visualized by incubating with nitroblue tetrazolium (0.33 m g / m l , GIBCO BRL, USA) and 5-bromo-4-chloro-3-indolyl-phosphate (0•165 m g / m l , GIBCO BRL, USA) in 100 mM Tris-HC1 (pH 9.5) containing 100 mM NaC1 and 50 mM MgC12. The 14 autopsied human brains employed in this study

1 200

2

were obtained within 2-32 h of death (Table 1). They included 7 cases of AD (aged 69-86 years) and 7 cases without neurological disorder (aged 58-85 years). The diagnosis of AD was confirmed in every case by the demonstration of large numbers of senile plaques and neurofibrillary tangles using Bielschowsky's silver stain. Small blocks of brain tissue of approximately 5 mm thickness were dissected and immediately fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 2 days. They were then transferred to a maintenance solution of 15% sucrose in 0.1 M phosphate buffer, pH 7.4. Sections were cut on a freezing microtome at 30 p.m thickness, collected in the maintenance solution, and stored until stained. The hippocampal formation, entorhinal cortex, middle temporal gyms and visual cortex areas were examined. Tissue from brains which were fixed in the same fixative but by whole brain immersion was also examined in preliminary experiments• Since the NF-KB staining of such tissue was somewhat variable, the results described here were confined to those obtained from tissue cut as a small block and immediately fixed. Immunostaining was performed on free-floating sections. Prior to staining, sections were treated for 30 min with 0.2% hydrogen peroxide to eliminate endogenous peroxidase activity. For all the procedures except the final DAB reaction, PBS containing 0.3% Triton X-100, pH 7.4

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K. Terai et al. / Brain Research 735 (1996) 159-168

(PBS-T) was used as a buffer solution. Following preincubation for 1 h at room temperature with a blocking solution containing 5% normal serum of the secondary antibody host in PBS-T, incubation with primary antibody was carried out for 3 days at 4°C. Sections were then treated with biotinylated secondary antibody diluted 1:1000 for 2 h at room temperature followed by incubation in the avidin-biotinylated HRP complex (ABC Elite system, Vec-

163

tor Lab., Burlingame, CA) diluted 1:4000 for 1 h at room temperature. Peroxidase labeling was detected by incubating with 50 m M Tris-HC1, pH 7.6 containing 0.01% 3,3"-diaminobenzidine (DAB, Sigma), 0.6% nickel ammonium sulfate, 0.05 M imidazole and 0.00015% hydrogen peroxide [15]. A purple reaction product appeared after about 15-45 min, at which time the reaction was terminated by transferring the sections to PBS-T. Sections were

Fig. 2. Representativeimmunostainingfor NF-KB in neuronal elements of Alzheimer's disease (A-E) in comparisonwith that of a control case (F,G) in the same region (hippocampalCAI area). In the Alzheimercases, the neuronalstainingis located in neurons(A), neurofibrillarytangles (B) and dystrophic neurites (C). Strongly stained neurons in the hippocampal CA1 area of Alzheimer (D,E) are similar in their localization and cellular shapes to faintly stained neurons in the same region of the control case (F,G). Scale bars = 50 p~m (A-C,E,G); 200 Ixm (D,F).

K. Terai et al. / Brain Research 735 (1996) 159-168

164

subsequently mounted on glass slides, dehydrated, and coverslipped with Entellan (Merck) with or without neutral red counterstaining.

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Western blots of human brain homogenates were performed to confirm the specificity of the antibody against the NF-KB p65 subunit. A prominent band with an esti-

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Fig. 3. In an Alzheimer case, comparison is shown between staining of neuronal elements immunoreactive for NF-KB in the hippocampal formation as a whole (A), C A 3 - 4 (13), dentate gyrus (C), subiculum (D) and entorhinal cortex (E). High power magnified regions (B E) are shown as boxed areas on lhe low power photomicrograph (A). Scale bars = 1 m m (A); 100 g m (B--D).

K. Terai et al./Brain Research 735 (1996) 159-168

mated molecular weight of 65 kDa was detected (Fig. 1A, lane 1). Three very weak bands of lower molecular weight were also observed. These may represent degradation products. Application of antibody preabsorbed with the synthesized peptide (lane 2) or omission of the first antibody (lane 3) resulted in a disappearance of the bands. An immunoabsorption immunohistochemical test using the synthesized peptide resulted in disappearance of any positive staining for NF-KB (cf. Fig. 1B and C). A similar result was obtained when the primary antibody was omitted. Decreasing the concentration of primary antibody significantly decreased the intensity of tissue immunostaining (data not shown). The antibody for NF-KB stained three types of structures: neuronal elements, glial cells and vascular walls (Table 1). The distribution patterns for each of these types is described below for the hippocampal formation (HIP), entorhinal cortex (ENT), middle temporal gyrus (MTG) and visual cortex (VIS) of AD and control cases. Weak and variable NF-KB staining of neurons was observed in control cases. By contrast prominent staining of neurons and their processes as well as neurofibrillary tangles and dystrophic neurites was seen in AD cases (Table 1 and Fig. 2). Enhanced staining was apparent in the HIP and ENT in all AD cases, in the MTG in six of the seven cases, and in the VIS in three of the six cases (Table 1). In some AD cases, stained neurons were sparse in areas

165

where positively stained neurofibrillary tangles were abundant (Table 1). At high magnification, some subcellular localization of the positive staining could be seen (Fig. 2 A - C ) . In neurons the staining was observed mainly in the cytoplasm but sometimes appeared to be in the nucleus as well (Fig. 2A). Positively stained neurofibrillary tangles were mainly intracellular but some weakly stained extracellular ones were also observed (Fig. 2B). Most positively staining dystrophic neurites were clustered inside senile plaques (Fig. 2C). Although only a few neurons were weakly stained in some control cases, they were similar in their shape and localization to the strongly stained neurons in AD cases (Fig. 2F,G). Neurofibrillary tangles and dystrophic neurites were not found in the control cases. Strongly stained neurons varied in their cellular shapes depending on the regions investigated. In CA1 to CA4 areas and the subiculum of HIP, large pyramidal neurons were preferentially stained (Fig. 3A,B,D). Granular neurons in HIP were stained moderately in only four of seven cases (Fig. 3C). In ENT, stained neurons were more round and smaller than those in HIP (Fig. 3E). In MTG (Fig. 4A,B) and VIS (Fig. 4C,D), large sized neurons were stained preferentially to smaller neurons. Moderately stained astroglia were observed near the pial surface in all regions of all the cases investigated (Table 1 and Fig. 5A,B). Vascular walls of small or

A

C Fig. 4. In an Alzheimer case, comparison is shown between neuronal elements immunoreactivefor NF-KB in the middle temporal gyrus (A,B) and visual cortex (C,D). High power magnified regions are shown as boxed areas on the low power photomicrographs. Scale bars = 500 Ixm (A,C); 100 p~m (B,D).

166

K. Terai et al. / Brain Research 735 (1996) 159-168

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K. Terai et al. / Brain Research 735 (1996) 159-168

medium size blood Vessels located in the gray matter of all specimens were weakly stained (Table 1 and Fig. 5A,C). A few microglia were stained in some specimens, but there was no definite difference between AD and control cases (Table 1). Positive cells were detected only in MTG and VIS and they were distributed homogeneously through the gray matter (Fig. 5D,E). Only in one case with infarction sites in the MTG and VIS were reactive astroglia strongly stained (Fig. 5F,G). In this study, we used an antibody against the p65 subunit of NF-KB which has been well characterized, and has been previously used in immunocytochemical studies [17,18,30]. AD-specific NF-KB staining was observed in neuronal elements, including morphologically intact neurons. Positive staining was seen mainly in the cytoplasm although it sometimes appeared also to occur in the nucleus. This accumulation of NF-KB in the cytoplasm may reflect high activation or overexpression of NF-KB. A recent study using a transgenic mouse strain overexpressing the NF-KB p65 subunit has indicated that cytoplasmic retention of overexpressed p65 by IKB is the major in vivo mechanism for controlling the excess NF-KB activity in long-term p65-overexpressing thymocytes [25]. The accumulation of NF-KB observed in our experiment may be caused by continuous induction of NF-KB expression by cytokines such as IL-2 which increase mRNA levels of NF-KB subunits [ 1]. The characteristic neuropathological features of AD are neurofibrillary tangles and senile plaques. These pathological elements are more prominently developed in the hippocampal formation, limbic system and temporal lobe in comparison with somatosensory, visual and motor areas of the cerebral cortex. Our neuropathological data from Table 1 show that the distribution of the neurofibrillary tangles and dystrophic neurites stained for NF-KB is similar to that of the general neuropathological damage. A recent report has suggested that abnormal glycation of tau in AD paired helical filaments causes activation of NF-KB non-enzymatically, which results in cytokine gene expression and the release of amyloid [3-peptide in neurons [39]. The neurofibrillary tangles and dystrophic neurites positively stained for NF-KB in the present study might result from continuous abnormal glycation during advancing AD pathological changes. Many studies have shown that multiple immune system components and cytokines are expressed in brain cells in AD, and that such expressions are related to the neurodegeneration of AD [14-16,28,29,34]. The expression of many of those molecules might possibly be induced by activation of NF-KB since it is a multiple gene inducer. Our data showed that the immunostaining of NF-KB neuronal elements was stronger in AD cases than in controls, but there was no difference in the staining of glial cells between AD and control cases. This suggests that NF-KB may not be a major inducer for gene expression of im-

167

mune-related molecules in glial cells of AD, but in neurons. However, another immunohistochemical study using the experimental autoimmune encephalomyelitis model in rats has demonstrated that NF-KB is activated in microglia of the spinal cord [9]. Recently, it has been demonstrated that one of the anti-inflammatory mechanisms of salicylates is the inactivation of NF-KB by preventing the degradation of IKB, an NF-KB inhibitor [11,37]. Nitric oxide, an endogenous molecule, has similar effects for the N F - K B / I K B system in human vascular endothelial cells [24]. Some neuronal populations have nitric oxide synthase producing nitric oxide [35,36]. Nitric oxide can possibly be used as a NF-KB inhibitor in these neurons. In fact, it has been shown that neurons containing nitric oxide synthase resist neuronal cell loss in advancing AD [7,12]. It may be important to regulate the activity of NF-KB for prevention of neurodegeneration in AD.

Acknowledgements This research was supported by grants from the Jack Brown and Family Alzheimer Disease Research Fund, the Alzheimer Society of British Columbia as well as donations from individual British Columbians.

References [1] Arima, N., Kuziel, W.A., Grdina, T.A. and Greene, W.C., IL-2-induced signal transduction involves the activation of nuclear NF-KB expression, J. Immunol., 149 (1992) 83-91. [2] Baldwin, A.S., Jr. and Sharp, P.A., Two transcription factors, NF-KB and H2TF1, interact with a single regulatory sequence in the class I major histocompatibility complex promoter, Proc. Natl. Acad. Sci. USA, 85 (1988) 723-727. [3] Blanar, M.A., Burkly, L.C. and Flavell, R.A., NF-KB binds within a region required for B-cell-specific expression of the major histocompatibility complex class II gene E~, Mol. Cell. Biol., 9 (1989) 844-846. [4] Blank, V., Kourilsky, P. and IsraEl, A., NF-~B and related proteins: Rel/dorsal homologies meet ankyrin-like repeats (Review), Trends Biochem. 8ci., 17 (1992) 135-140. [5] Ernst, M.K., Dunn, L.L. and Rice, N.R., The PEST-like sequence of IKBc~ is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers, Mol. Cell. Biol., 15 (1995) 872-882. [6] Griffin, G.E., Leung, K., Folks, T.M., Kunkel, S. and Nabel, G.J., Activation of HIV gene expression during monocyte differentiation by induction of NF-KB, Nature, 339 (1989) 70-73. [7] Hyman, B.T., Marzloff, K., Wenniger, J.J., Dawson, T.M., Bredt, D.S. and Snyder, S.H., Relative sparing of nitric oxide synthase-containing neurons in the hippocampal formation in Alzheimer's disease, Ann. Neurol., 32 (1992) 818-820. [8] IsraEl, A., Le Bail, O., Hatat, D., Piette, J., Kieran, M., Logeat, F., Wallach, D., Fellous, M. and Kourilsky, P., TNF stimulates expression of mouse MHC class I genes by inducing an NFKB-Iike enhancer binding activity which displaces constitutive factors, EMBO J., 8 (1989) 3793-3800. [9] Kaltschmidt, C., Kaltschmidt, B., Lannes-Vieira, J., Kreutzberg,

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[10]

[11] [12]

[13]

[14]

[15]

[16]

[17]

[18]

[19] [20]

[21]

[22]

[23]

[24]

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