Yan Zhan1, 2, Dihui Ma2, Yu Zhang2, Ming Chang2, Linsen Hu2

1Institute of Genetics and Cytology, School of Life Sciences, Northeast Normal University, Changchun  130024, Jilin Province, China
2Department of Neurology, First Hospital, Jilin University, Changchun  130021, Jilin Province, China

Yan Zhan☆, Doctor, Asso-ciate chief physician, Institute of Genetics and Cytology, School of Life Sciences, Northeast Normal University, Changchun  130024, Jilin Province, China; Department of Neurology, First Hospital, Jilin University, Changchun  130021, Jilin Province, China

Corresponding author: Yan Zhan, Institute of Genetics and Cytology, School of Life Sciences, Northeast Normal University, Changchun  130024, Jilin Province, China; Department of Neurology, First Hospital, Jilin University, Changchun  130021, Jilin Province, China
zhanyan2004@sina.com

Supported by: the Jilin Provincial Technology Development Foundation, No. 200505204*, 200705129*; the Postdoctorate Foundation from Northeast Normal University, No. 111258000*

www.crter.cn
www.nrronline.org

doi:10.3969/j.issn.1673-5374.2010.11.004

Abstract
BACKGROUND: To date, no drugs are able to halt the progression of Alzheimer’s disease (AD). Neural stem cells (NSCs) transplantation has been widely used to treat AD, but the mechanism of AD treatment remains unclear.
OBJECTIVE: To observe changes in protein and factors in the hippocampus and frontal lobe of AD rats following NSCs transplantation, and to understand mechanism of action of NSCs transplantation in AD treatment.
DESIGN, TIME AND SETTING: A randomized, controlled animal study was conducted at the First Clinical Hospital, Jilin University, China from July 2007 to March 2009.
MATERIALS: NSCs were harvested from the hippocampus of 10 E16 Wistar rats.
METHODS: A total of 57 male adult Wistar rats were equally and randomly divided into normal control, AD model and NSCs groups. AD models were established in the AD model and NSCs groups by bilateral removal of hippocampus. At 2 weeks postsurgery, NSCs were transplanted into the hippocampus of rats from the NSCs group.
MAIN OUTCOME MEASURES: Protein levels were measured in the hippocampus of rats from normal control, NSCs and AD model groups using proteomics. Expression of choline acetyl transferase mRNA, glial fibrillary acidic protein and S100β was measured in the hippocampus and frontal lobe of rats using in situ hybridization and immunohistochemistry.
RESULTS: Expression of choline acetyl transferase mRNA, heat shock protein 70, heat shock protein 90, F-actin and actin was significantly higher in the NSCs group compared with AD model group. Glial fibrillary acidic protein and S100β expression was less in the NSCs group compared with AD model group.
CONCLUSIOIN: NSCs implanted into the brain may generate new neural cells, which can relieve damage to the cholinergic system and resist apoptosis. NSCs transplantation plays a protective role in the cholinergic system in the AD rats to some extent.
Key Words: neural stem cells; Alzheimer’s disease; choline acetyl transferase; glial fibrillary acidic protein; S100β; proteomics

INTRODUCTION
   
Alzheimer’s disease (AD) is characterized by a progressive loss of neurons which leads to dementia[1-2]. At present, the pathogenesis of AD is still unclear. Several hypotheses of AD currently exist. For instance, in the “cholinergic hypothesis” it is assumed that cognitive and memory impairments are caused by a cholinergic deficit[3]. By this hypothesis, cholinergic neurons in the nucleus basalis of Meynert and the hippocampus are most affected in AD. With progression of the disease, the levels of the neurotransmitter acetylcholine decrease and subsequent neuronal loss leads to widespread cell death.
Current drug treatments are aimed at raising acetylcholine levels in cholinergic areas of the brain[4]. Drugs such as cholinesterase inhibitors (tacrine, donepezil, rivastigmine and galantamine) are centrally active and have been shown to improve memory and cognition in some patients with mild to moderate AD[5]. However, none of these drugs halt progression of the disease, and new therapies for AD are needed. Recently, much attention has been focused on the relevance of hippocampal neurogenesis to the pathophysiology and treatment of memory loss[6-7]. Altered hippocampal neurogenesis may also play a pathophysiological role in neurodegenerative disorders such as AD[8-10]. Heat shock proteins and cytoskeletal proteins are regulators of anti-apoptotic mechanisms in cells. Heat shock protein 70 has significant effects on myocardial damage. More experiments are needed to prove whether heat shock protein 70 content or function changes in the brain of experimental animals. Studies of protein changes are especially needed in rat  
models of AD after neural stem cells (NSCs) transplantation.
Stem cells have been widely used to treat neurodegenerative diseases in animal experiments[11-14]. NSCs can self-renew and differentiate into major classes of central nervous system cells, such as neurons, astrocytes and oligodendrocytes[15]. Many studies have addressed NSCs treatment of AD. However, most of these focused on in vitro culture data and the study of behavior and pathology in AD models that received NSCs transplantation. In this experiment, we analyzed AD rat brains that received fetal NSCs using proteomics, in situ hybridization, and immunohistochemistry to explore protein expression in the hippocampus and changes in the expression of choline acetyl transferase (ChAt) mRNA, glial fibrillary acidic protein (GFAP) and S100β in the hippocampus and frontal lobe of rats from each group, the results of which may provide valuable clues to explain protective effects of transplanted NSCs on AD rat brains.

MATERIALS AND METHODS

Design
A randomized, controlled animal study.
Time and setting
Experiments were performed at the First Clinical Hospital, Jilin University, China from July 2007 to March 2009.
Materials
Main reagents and equipment are listed as follows:

A total of 10 clean, E16 Wistar rats and 57 adult, male, Wistar rats, weighing 240–250 g, were obtained from the Experimental Animal Center of Jilin University in China (license no. SCXK (Ji) 2007-0003). Protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[16].
Methods
Preparation of NSCs
Cell culture: Time-pregnant female Wistar rats at embryonic day 16 were deeply anesthetized with chloral hydrate (0.33 mL/100 g, intraperitoneally) and a midline incision was made to expose the embryos. The hippocampus of embryonic rats was removed under a dissecting microscope and mechanically dissociated using a fire-narrowed pipette and centrifuge. The resulting cell suspension was plated in the presence of 20 μg/L EGF and 10 μg/L bFGF in DMEM/F12 (1: 1 medium) and placed in a humidified incubator at 37 °C and 5% CO2. Subsequently, medium supplemented with EGF (40 μg/L) and bFGF (20 μg/L) was changed every 3 days. These conditions promote formation of neurospheres from floating cultures of single cells. Proliferation of primary cells reached a peak after 6 days. The neurospheres were collected, and the cell concentration was adjusted to about 5 × 106/μL.
Cell identification: from 4 to 12 days after plating, cells were centrifuged, spotted, fixed, and identified by Nestin, a NSC specific marker[6]. Before transplantation, 5-BrdU (5 μmol/L) was added to the culture medium. After 24 hours of incubation, NSCs were collected, and the cell concentration was adjusted to about 5 × 105/μL.
Grouping and experimental model
A total of 57 male adult Wistar rats were equally and randomly divided into normal control, AD model, and NSC groups. Four rats from each group were used in the proteome experiment, and 15 rats for histomorphological examination.
The rat AD model was established as previously described[17]. Rats were intraperitoneally anesthetized with chloral hydrate. Under aseptic conditions and with the aid of an encephalon stereotaxic apparatus, the rat dura mater was opened, and bilateral hippocampi were transected by a double-face knife that was lowered into the brain (anterior–posterior, 1.8; lateral, 2.5; and dorsoventral, 5.5), moved laterally to 4.0 mm bilateral to the midline and retracted at this position[17-19]. Hippocampi of Wistar rats were transected bilaterally to simulate the impairment of cholinergic neurons in AD rats by lesioning the septohippocampal pathway. Behavior of AD rats was measured using the Morris water maze[17, 20].
NSCs transplantation
At 2 weeks postsurgery, rats in the NSCs and AD model groups received homotransplants with NSCs (2.5 × 105 cells in 5 μL) and sterile physiological saline (5 μL), respectively, utilizing 20 μL microsyringes. The injection site (anterior 4.5 mm, lateral ± 3.5 mm, ventral 3.6 mm) was selected using Paxinos’s atlas as a reference[17-19]. 5 to 6 μL of NSCs or sterile physiological saline were slowly injected stepwise 1 to 2 mm rostral to the lesion site (hippocampus) over 10 minutes to prevent oozing of fluid along needle tract. During surgery, rectal temperature was monitored and maintained at (37.0 ± 0.5) °C with a heating pad (homemade in our lab). After grafting, the dural sheath and skin were sutured, followed by intramuscular injection of penicillin (dose: 6 mg/g, twice daily.) to prevent postoperative infection.
Proteomics
Sample preparation: at 4 weeks after transplantation, four rats from each group were deeply anesthetized. The hippocampus tissue was quickly removed, weighed on an analytical balance and washed twice with ice-cold phosphate buffered saline (PBS). Lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 30 mmol/L Tris (pH 8.8), containing 1% protease inhibitors and nuclease mix) was added to lyse the tissues. The supernatants were collected after centrifugation at 25 000 r/min for 30 minutes. The protein samples were cleaned up and quantified using Clean-up kit and two dimensional electrophoresis Quant kit in accordance with the manufacturer’s instructions.
Two-dimensional difference gel electrophoresis: Samples (following the protocols of 50 μg protein per sample recommended by manufacturer) from each group were minimally labeled with fluorescent dyes (density of sample: 5–10 g/L; protein: fluorochrome, 50 μg:      400 pmol). After incubating on ice for 30 minutes, the reaction was terminated by adding 1 μL of 10 mmol/L lysine. Cy3-, Cy5- and Cy2-labeled samples and internal standards were pooled and rehydrated in rehydration buffer (8 mol/L urea, 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 0.4% immobilized pH-gradient buffer and 0.28% dithiothreitol) to equal volumes. The samples were loaded on an immobilized pH-gradient (IPG) strip (24 cm, pH 3–10, linear) for isoelectric focusing on an IPGphor. The protocol was as follows: 20 °C, 30 V for 12 hours (rehydration); 500 V for 3 hours; grad 1 000 V for 1 hour; grad 8 000 V for 1 hour; and 8 000 V for 3 hours. After isoelectric focusing, the strips were equilibrated in equilibration solution (50 mmol/L Tris-HCl, pH 8.8,      6 mol/L urea, 30% v/v glycerol, 2% w/v sodium dodecyl sulfate and 1% w/v dithiothreitol) for 15 minutes and later in a similar solution with dithiothreitol replaced by 4%  iodoacetamide (w/v) for an additional 15 minutes. Subsequently, the IPG strips were loaded on 12.5% polyacrylamide gels in Ettan DALT Six system for electrophoresis at 1.5 W/gel overnight until the bromophenol blue dye front reached the bottom of the gel.
Image analysis: fluorescent images were collected with a Typhoon 9400 scanner at a resolution of 100 μm. Matching, quantification and statistics were carried out with DeCyder Differential in-Gel Analysis software and images were checked manually to eliminate artifacts. Comparisons of protein expression in two-dimensional images were performed. The pixel volume of each spot was calculated, normalized and compared between groups. Every gel was matched to an internal standard image of itself to minimize gel–gel variance. To judge differences in protein expression, 1.3-fold change was considered significant, only proteins with a 1.3-fold increase or decrease were used. Three-dimensional simulation of the protein spot was provided by DeCyder software analysis, which was more objective than the conventional way. Gels were fixed in 20% trichloroacetic acid overnight and stained with PhastGel Blue. Preparative gels from different groups were all made with 450 μg protein under the same conditions. Spots of interest were excised from the difference electrophoresis analytic gels and preparative gels with the help of an Ettan Picker for further identification (samples were marked with a 1, 2, 3, 4 or 5).
Protein identification by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS): Gel plugs picked were digested with trypsin automatically on an Ettan Digester. First, they were destained with 50 mmol/L NH4HCO3-50% methanol, and then dehydrated with 50% acrylonitrile-0.1% trifluoroacetic acid. After the gel plugs had dried completely, trypsin solution (20 mg/L in 20 mmol/L ammonium bicarbonate) was added and the samples were digested at room temperature overnight. The extracted peptides were removed, dried and resuspended in 50% acrylonitrile-0.1% trifluoroacetic acid. Equal volumes of sample and cyano-4-hydroxy cinnamic acid were spotted and mixed on the MALDI-TOF-MS target slides by Ettan Spotter.
Peptide extracts were analyzed on a MALDI-TOF-MS in positive ion reflection mode. The accelerating potential was 20 kV with eight shots per second. Trypsin autodigestion peaks were used as internal calibration and human adrenocorticotropic hormone (19–39) and Ang III as external controls. Peptide mass fingerprint spectra were compared against National Center for Biotechnology Information using the ProFound search engine[21-23] and the rattus species. The requirements for identification were that the expectation value (chance of misidentification) was less than 0.05 and the coverage (the ratio of the protein sequence covered by the matched peptides) was more than 20%. The results were further confirmed in the Swiss-Prot protein database (us.expasy.org/sprot).
Tissue processing for morphology detection
At 4 weeks after transplantation, 15 rats from each group were deeply anesthetized with chloral-hydrate      (0.33 mL/10 g, intraperitoneally) and perfused through the aorta with 200–250 mL of 0.9% physiological saline, followed by 300 mL ice-cold 4% paraformaldehyde in  0.1 mol/L phosphate buffer, pH 7.4. The brains were removed from the skull and postfixed in 4% paraformaldehyde by immersion for 24 hours, and then transferred into gradient alcohol, immersed in paraffin, and embedded after dimethyl benzene was used. The brain samples were cooled by solid CO2 and cut into 40-μm-thick coronal sections on a freezing cryostat for further processing, and then were stored at –80 °C until use. A total of 10 slices of hippocampus were made in the same position from each sample. Five slices were used in immunohistochemistry experiments and five slices were used for in situ hybridization.
Immunofluorescence staining
For immunohistochemical detection of BrdU labeling[24-25], brain sections were fixed in 4% paraformaldehyde, pretreated to denature DNA in 50% formamide-2×saline-sodium citrate buffer at 65 °C for   2 hours, and then incubated at 37 °C for 30 minutes in  2 mol/L hydrogen chloride. Sections were rinsed for  10 minutes at 25 °C in 0.1 mol/L boric acid, pH 8.5. Sections were then incubated in 0.3% H2O2 for 30 minutes and blocked with 10% normal horse serum for 30 minutes. Sections were incubated overnight with mouse anti-rat BrdU monoclonal antibody (1: 200) diluted in PBS with 2% horse serum and 0.3% Triton X-100, and incubated with biotinylated horse anti-mouse IgG (1: 200) for 1 hour at room temperature, and then mounted and observed using fluorescence microscope.
Immunohistochemical analysis
Immunohistochemistry was performed on free-floating sections of the grafted brain sections as follows. Endogenous peroxidase was quenched with 3% hydrogen peroxide, brain sections were pre-incubated in 1% bovine serum albumin/0.2% Triton X-100/0.1 mol/L PBS for 30 minutes at room temperature. The sections were then incubated overnight at 4 °C with primary antibody (rabbit anti-rat GFAP 1: 200 or rabbit anti-rat S100β) in 0.1 mol/L PBS supplemented with 0.5% bovine serum albumin. Following rinsing in 0.5% bovine serum albumin/0.1 mol/L PBS, the sections were incubated for 1 hour at room temperature in biotinylated anti-rabbit secondary antibody (1: 200) and then rinsed in 0.5% bovine serum albumin/0.1 mol/L PBS. Sections were transferred to an avidin-biotin complex in 0.5% bovine serum albumin/0.1 mol/L PBS for 1 hour. The secondary antibodies were visualized with an avidin-biotin-peroxidase complex, using 0.05% 3, 3’-diaminobenzidine as a chromogen in the presence of 0.03% H2O2 in PBS for 5–10 minutes. After stopping the reaction by rinsing in 0.1 mol/L PBS, the sections were mounted on gelatin-coated slides. A total of 3 slices of hippocampus and 3 slices of frontal lobe of each rat were selected as samples. Positive cells in 100 squares per visual field were countered via ocular micrometer of an optical microscope and analyzed by statistics.
In situ hybridization
Before in situ hybridization, sections were digested in 1 mg/L protease K, fixed in 4% buffered paraformaldehyde for 15 minutes, rinsed three times with 0.1 mol/L PBS, and placed in 0.1 mol/L triethanolamine containing 0.25% acetic anhydride for 10 minutes. Subsequently, prehybridization was performed in hybridization buffer (hybridization buffer: 50% formamide, 10% dextran sulfate, 2% sodium dodecyl sulphate, 100 μg/mL salmon sperm DNA, 5 × saline sodium citrate) at 55 °C for 2 hours. Denatured probes (0.5 g/L hybridization buffer) were hybridized for 12 hours at 55 °C. Probe subsequences are listed in Table 1.

Statistical analysis
All data were statistically analyzed using SPSS 11.0 software (SPSS, Chicago, IL. USA) and were analyzed utilizing t-test and analysis of variance. Student t-tests were employed to compare between groups. A value of  P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of laboratory animals
All 57 rats were included in the final analysis without loss.
NSCs transplantation (Figure 1)

Some fluorescently labeled NSCs and a number of nestin-positive cells were visible in the brain of the NSCs group (Figure 1).
Proteomic changes in the hippocampus
Protein spot 3 identified as GFAP by MALDI-TOF MS was higher in AD rats than that in normal controls. Spots 1 and 2 were identified as heat shock proteins and spots 4 and 5 as cytoskeletal proteins were all lower in the AD model group than those in the normal control group. However, these protein spots were normal in the NSCs group (Table 2, Figure 2). Peptide mass fingerprinting of heat shock protein 90 is shown in Figure 3.

Expression of ChAt mRNA in different experimental rats
In this study, in situ hybridization showed that ChAt mRNA-positive cells were visible in frontal cortex and hippocampus in all groups after NSCs transplantation.  The number of ChAt mRNA-positive cells in the AD model group differed significantly from the NSC and normal control groups (P < 0.01) (Figure 4).

Changes in GFAP and S100β in experimental rats
GFAP and S100β positive cells were detected in the frontal cortex and hippocampus in all groups. The numbers of GFAP and S100β positive cells in the NSCs group were significantly lower compared with the AD model group (P < 0.01) (Figures 5, 6).

DISCUSSION

Bilateral hippocampal lesions are used in rats to produce an animal model of AD according to the cholinergic hypothesis[3, 18]. The degree of deficit is proportionally related to the number of cholinergic neurons that degenerate, which is closely associated with learning and memory function. ChAt has been used as a marker of cholinergic neurons for many years[26-27]. In addition, cytoskeletal destruction might be a critical mechanism of cell dysfunction and neurofibrillary tangles formation in AD brain[28]. Structural changes in synapses are also a focus of studies addressing learning and memory. The main components of cytoskeletal proteins, F-actin and actin form the basis of dendritic spine regulation, movement, growth and plasticity as they are integrated and depolymerized.
Since mechanical lesions can cause astrocytic activation, people have speculated that astrocyte response plays a key role in the pathophysiological processes of this AD model. As an EF-hand Ca2+ binding protein, S100β is primarily produced by astrocytes in the central nervous system. Increased levels of S100β has been observed in patients suffering from chronic neurodegenerative disorders such as Alzheimer’s disease[29-31]. S100β as well as that of GFAP, the major component of neuroglia filaments which is mainly localized in fibrous astrocytes[32]. A previous study suggested that the outcomes of some nervous system diseases could be initially assessed by GFAP and S100β expression[33]. The present study demonstrates that the number of surviving cholinergic neurons, F-actin, and actin are significantly greater in the NSCs group compared with the AD model group. Transplanted NSCs may generate new neurons that may compensate for the cholinergic deficit. After cells are transplanted in vivo, their differentiation is strongly influenced by environmental signals and cellular deficiencies in sites of central nervous system injury or disease[34-35].
In this study, the numbers of GFAP and S100β-positive cells were significantly lower in the NSCs than the AD model group. Following transplantation into brain, NSCs can differentiate into new neural cells that can resist apoptosis and produce new neural nets that contain nerve conduction channels and improve memory and cognition of AD rats[17]. This process could decrease GFAP and S100β expression.
Heat shock protein 70 and heat shock protein 90 expression changed between experimental groups. One or more of the many activities ascribed to heat shock proteins may mediate their beneficial effects, including refolding denatured proteins and preventing unfolded and damaged proteins from aggregating, or by a direct anti-apoptotic mechanism. Understanding the aspects of heat shock protein function required for protection will help us identify pathological processes that contribute to cell death and allow future work on neuroprotection to target identified pathological mechanisms. Several studies have shown a correlation between heat shock protein 70 and decreased protein aggregation and protection from AD[36-39]. Heat shock protein 90 can prevent the aggregation of unfolded proteins and cooperate with the heat shock protein 70/ heat shock protein 40 chaperone system in the adenosine triphosphate-dependent refolding of unfolded model proteins[40]. However, there has been no conclusive report on the function of heat shock protein 90 in AD.
During proteomic mass spectrometry, a single protein could be identified in several discrete spots, presumably representing protein modifications (such as spots 1 and 2 in this study). This suggests that posttranslational modifications consisting of addition or removal of a small charged moiety occurs. We analyzed several protein spots, several others (GFAP, heat shock protein 90, heat shock protein 70, F-actin, actin) and other protein spots will be identified in follow-up trials.
NSCs implanted into the brain may generate new neurons, which can survive and produce series of new neural networks. The results of this study indicate that NSCs can repair lesioned hippocampus and may provide some beneficial information based on reliable experiment results. NSC-based neuroreplacement therapies may have a bright future in the treatment of AD.

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 (Edited by Li LX/Qiu Y/Song LP)