Antibodies were supplied by Santa Cruz Biotechnology Inc. (ErbB4, sc-283, sc-8050; p-ErbB4, sc-33040) (Santa Cruz, CA, USA) and COVANCE (beta-amyloid, SIG-39320) (Emeryville, CA, USA). Vectashield (H-1000), biotinylated anti-rabbit IgG (BA-1000) and biotinylated anti-mouse IgG (BA-2001) were supplied by Vector Laboratories (Burlingame, CA, USA). Hoechst 33342 (bis-benzimide H33342 trihydrochloride, 14533) and DAB (3,3'-diaminobenzidine tetrahydrochloride, D-5905) were supplied by Sigma Aldrich (St. Louis, MO, USA).

Fourteen age-matched control brains with no clinical or neuropathologic evidence of AD were obtained within 24 postmortem hours from the cadavers of the Department of Anatomy and Neuroscience, School of Medicine at Eulji University. Consent was given for brain donation by next-of-kin in all cases, and a postmortem examination was conducted by the Anatomy laboratory according to standardized protocols. All experimental procedures were performed in accordance with 'The Guidelines of the Institutional Review Board (Ethics Committee) at Eulji University.' A total of fourteen hemispheres of brains obtained by autopsy were fixed in 10% neutral buffered formalin. The fixed hemispheres were cut in coronal plane, and blocks were selected from 10 to 15 brain areas for pathologic diagnosis and a comprehensive evaluation of the neurodegenerative process. They were prepared according to routine histological procedures for paraffin section. The fifteen Alzheimer brains were generously gifted from Dr. Roger A. Brumback and were diagnosed by him (Department of Pathology, School of Medicine, Creighton University, Omaha, NE, USA). All brains were processed according to same protocols. The sections were cut in coronal plane at 8 µm for immunohistochemical study or 20 µm for confocal microscopic immunofluorescence study, and then mounted on poly-L-lysine coated slides. The sections were deparaffinized and hydrated according to routine graded xylene-alcohol methods. Hematoxylin and eosin (H&E) and Luxol fast blue/cresyl violet (LFB-CV) stains were done for general histopathology and modified silver stain by Campbell et al. [24] for detecting the senile plaque and Gallyas stain [25] for NFT were employed. The modified Campbell's silver staining method is a combination of the Campbell and Gallyas stain. The technique was modified in our laboratory for the detection of brain amyloid and NFT in humans. Briefly, deparaffinized slides are placed into a pyridine silver solution for the induction of nucleation sites, which is followed by incubation in a Gallyas' physical developer solution for 8-10 minutes. The sections are then checked under the microscope. The Gallyas developer is composed of three solutions. Solution A contains 50 g of sodium carbonate dissolved in 1,000 ml of distilled water (DW). Solution B contain 2 g of ammonium nitrate, 2 g of silver nitrate, and 10 g of tungstosilicic acid dissolved in 1,000 ml of DW. Solution C is the same as the B solution, except 7 ml of 40% formol is added. All solutions are mixed prior to the staining process (8 : 10 : 2 vol %; sol. A : sol. B : sol. C). The process is then fixed in 3% sodium thiosulfate solution in order to remove the unreacted silver and stop the silver impregnation and then rinsed in tap water for 30 minutes. The detailed data of each brain is summarized in Table 1.

The mouse model used in this study was the double transgenic APPSwe/PS1 (B6C3-Tg (APPswe, PSEN1dE9)85Dbo/J) from Jackson Laboratories (Bar Harbor, Maine). APPSwe is the Swedish mutation of the amyloid precursor protein, and PS1 is the mutant form of human presenilin 1. The sixteen-month-old wild type and transgenic mice were anesthetized with a mixture (0.01 mg/g body weight, i.p.) of ketamine (10 mg/ml) and rumpun (1 mg/ml). Mice were subjected to intracardiac perfusion-fixation using 4% paraformaldehyde dissolved in phosphate buffered saline (PBS), pH 7.4. Brains were removed and then prepared according to routine histological procedures for paraffin sectioning. The sections, cut 6 µm in thickness, were deparaffinized and hydrated according to routine graded xylene-alcohol methods. H&E stain was done for general histopathology.

In order to retrieve antigenecity, dewaxed sections were boiled within 0.1 mol/L citrate-buffered saline (pH 6.0) for 10 minutes. After cooling down for 30 minutes, the sections were rinsed in PBS. The endogenous peroxidase was quenched by 1% hydrogen peroxide in 10% methanol for 30 minutes. After two changes of PBS-T (0.1% Triton X-100 in 0.1 mol/L PBS, pH 7.6) washing for 5 minutes respectively, the sections were blocked for 1 hour in blocking solution (5% host serum+1% bovine serum albumin in PBS-T) and incubated in primary antibody (1 : 200, anti-ErbB4 and 1 : 200, APP) at 4℃ overnight. After PBS-T rinses, the sections were incubated in a biotinylated secondary antibody for 1 hour at room temperature (RT). After PBS rinsing and an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories) treatment for 1 hour at RT, the sections were developed for 5 minutes in a 0.05% DAB solution. Images were captured with a Axiocam digital camera (MRC, Carl Zeiss Inc., Göttingen, Germany) attached on the Olympus AX70 microscope (Olympus Co., Tokyo, Japan).

Immunofluorescence was performed as previously described [26]. In brief, the prestaining process was the same as above. Normal horse serum was then applied for 1 hour to block nonspecific background staining. Then sections were incubated with avidin-biotin blocking solution (Vector Laboratories) and incubated overnight at 4℃ in blocking solution containing rabbit anti-ErbB4 (1 : 50). Following this, they were washed and incubated with the appropriate secondary biotinylated antibody and after rinsing, the sections were incubated with FITC avidin D. The sections were then incubated with Hoechst 33342 for 30 minutes and mounted. The images were visualized using the ErbB4 with FITC (green) fluorescence and Hoechst 33342 with UV (blue) fluorescence on the same sections, using a LSM 510 meta system (Zeiss LSM 510 laser scanning microscope, Carl Zeiss Inc.). To reduce auto fluorescence, the brain sections were incubated in 10% Sudan black B solution for 5 minutes, and then rinsed with disilled, deionized water (DW) and mounted with Vectashield [27].

Western blotting was carried out as previously described [17]. Briefly, cells were lysed in a modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium-deoxycholate, 1 mM PMSF, 1 mM EDTA, 1 µg/ml aprotinin, lepeptin, and pepstain protease inhibitors). Protein was then resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes and subsequently blocked with TBS containing 5% nonfat powdered milk and 0.05% Tween 20 for 1 hour. Following this, membranes were incubated overnight at 4℃ with primary antibodies and developed using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence system (Amersham Pharmacia, Buckinghamshire, UK).

Within each region from three tissue sections, two images were captured at evenly spaced intervals to represent the entire anatomic area of interest. To determine the optical density of the immunoreactive cell, we used customized Image J software (NIH). Statistical analysis was conducted with Graph Pad Prism (GraphPad software, San Diego, CA, USA). Data were presented as means±standard error of the mean of three or more independent experiments. For multiple group comparisons, statistical differences were calculated using one-way ANOVA followed by Bonferroni's test. For comparison of means from the same group of cells, a Student's paired t-test was used. Values of P<0.05 were considered significant.

All brains (control and AD) were assigned Braak scores using neuropathological staging by Braak and Braak [28]. The two braak groups are referred to as age-matched control (no pathologic sign) or AD (Braak stage I-II) referring to the extent of NFT and senile plaque in the hippocampus. Postmortem examination confirmed the diagnosis of AD given the because the presence of abundant modified Campbell silver stained amyloid deposits and NFT; H&E and LFB-CV staining also demonstrated marked cell loss (Fig. 1A, B).

Previous biochemical studies have shown that NRG1 exerts neuropreotective effects in Alzheimer's cell model via ErbB4 [29]. However, it is not clear ErbB4 immunoreactivity was changed in the neurons of AD brain. Since ErbB4 immunoreactivity is associated with neuritic plaques [30], we hypothesized that ErbB4 immunoreactivity was changed in the neuron of hippocampal fields of AD brains. As expected, ErbB4 immunoreactivity was significantly increased in the pyramidal neurons of the CA1-2 transitional field of AD brains as compared to age-matched controls (Fig. 2B, left column and 2C). As shown in Fig. 2B (right column) and 2C, phospho-ErbB4 immunoreactivity was also more increased in the same fields. At high magnification, the signal was revealed in pyramidal neurons. ErbB4 immunoreactivity was found primarily in pyramidal neurons; a subset of smaller, multipolar neurons was also observed. ErbB4 immunoreactivity was further confirmed using confocal immunofluorescence on the same section. We found that the intensity of immunoreactivity of ErbB4 was higher in the neurons of the CA1-2 transitional field of AD brains as compared to age-matched controls (Fig. 2D, E). These results are in agreement with observations above that detected with immunostaining method. This suggests that ErbB4 mediated signaling is involved in AD processing.

AD is strongly correlated with neuronal cell loss in the limbic structures, such as hippocampus and the amygdala, and the associated regions of cerebral cortex. To further determine whether ErbB4 immunoreactivity was changed in the corticomedial nucleus amygdala of AD brains. As shown in Fig. 3A and D, ErbB4 immunoreactivity was significantly increased in comparison to age-matched control. The cholinergic neurons of the human basal forebrain (BFCN) undergo selective loss in neurodegenerative disorders of the elderly, particularly AD [31]. Thus, we investigated the signals of ErbB4 in the neurons of BFCN. As shown in Fig. 3B and E, ErbB4 immunoreactivity was significantly increased in comparison to age-matched control. Unlike limbic stuructures, loss of pyramidal neurons in the laminae III and V of the superior fornatal gyrus was not severe in early AD. As AD progresses, the pyramidal neurons with apoptotic morphology (dark degeneration) appeared more in the layer V. As shown in Fig. 3C and F, ErbB4 immunoreactivity more intense in the pyramidal neuron of AD.

Double transgenic mice express a Swedish mutation (APP K₅₉₅N/M₅₉₆L) and a mutant human presenilin 1 (PSEN₁dE₉). APP/PS1 double transgenic mice develop numerous neuritic plaques throughout the hippocampus, entorhinal cortex (data not shown), and cerebral cortex (Fig. 4A). In cerebral cortex and hippocampus of APP/PS1 double transgenic mice, ErbB4 immunoreactivity significantly increased in comparison to age-matched wild type control (Fig. 4A, B). Consistent with the ErbB4 expression observed in human AD, the intensity of immunoreactivity of ErbB4 was higher in the neuron of AD. To assess whether the level of expression of ErbB4 is altered in APP/PS1 double transgenic mice, lysates were prepared from the cerebral cortex and hippocampus. A protein of the expected 185 kD size was readily detected in immunoblots with the anti-ErbB4 antibody (Fig. 4C), but there were no differences in the level of ErbB4 expression in age-matched wild type control versus APP/PS1 double transgenic mice, in agreement with earlier studies [30].